In vitro fusion of lung lamellar bodies and plasma membrane is augmented by lung synexin.

Lamellar bodies of lung epithelial type II cells undergo fusion with plasma membrane prior to exocytosis of surfactant into the alveolar lumen. Since synexin from adrenal glands promotes aggregation and fusion of chromaffin granules, we purified synexin-like proteins from bovine lung cytosolic fraction, and evaluated their effect on the fusion of isolated lamellar bodies and plasma membrane fractions. Synexin activity, which co-purified with an approx. 47 kDa protein (pI 6.8), was assessed by following calcium-dependent aggregation of liposomes prepared from a mixture of phosphatidylcholine:phosphatidylserine (PC:PS, 3:1, mol/mol). Lung synexin caused aggregation of liposomes approximating lung surfactant lipid-like composition, isolated lamellar bodies, or isolated plasma membrane fraction. Lung synexin promoted fusion only in the presence of calcium. It augmented fusion between lamellar bodies and plasma membranes, lamellar bodies and liposomes, or between two populations of liposomes. However, selectivity with regard to synexin-mediated fusion was observed as synexin did not promote fusion between plasma membrane and liposomes, or between liposomes of surfactant lipid-like composition and other liposomes. These observations support a role for lung synexin in membrane fusion between the plasma membrane and lamellar bodies during exocytosis of lung surfactant, and suggest that such fusion is dependent on composition of interacting membranes.     

Binding of annexins to lung lamellar bodies and the PMA-stimulated secretion of annexin V from alveolar type II cells

To identify lung lamellar body (LB)-binding proteins, the fractions binding to LB-Sepharose 4B in a Ca(2+)-dependent manner from the lung soluble fractions were analyzed with Mono Q column. Four annexins (annexins III, IV, V, and VIII) were identified by partial amino acid sequence analyses as the LB-binding proteins in the lung soluble fractions. A control experiment using phospholipid (phosphatidylserine/phosphatidylglycerol/phosphtidylcholine) liposome-Sepharose 4B revealed that annexins III, IV and V were the Ca(2+)-dependent proteins binding to the column in the lung soluble fractions, while annexin VIII was not detected. Thus, annexin VIII might preferentially bind to LB. On the other hand, the only Ca(2+)-dependent LB-binding protein identified in the bronchoalveolar lavage fluids was annexin V. It was further demonstrated that annexin V was secreted by isolated alveolar type II cells from rats and that the secretion was stimulated by the addition of phorbol ester (PMA), a potent stimulator of surfactant secretion. The PMA-dependent stimulation of annexin V was attenuated by preincubation with surfactant protein-A (SP-A), a potent inhibitor of surfactant secretion. As LB is thought to be an intracellular store of pulmonary surfactant, which is secreted by alveolar type II cells, annexin V is likely to be secreted together with the lamellar body.     

A mode of formation of tubular myelin from lamellar bodies in the lung

A mechanism is suggested by which the membranes of lamellar bodies are converted to tubular myelin (TM) in the lung. It is argued that a simple corrugation of the membranous sheets can produce the TM formation. Such corrugation would occur in response to simple stresses acting on the lamellar body membranes. The intersections of the tubular figures are formed by fusion of adjacent corners in the corrugations. This results in a more stable hydrophobic bonding of phospholipid molecules. Strong supportive evidence for the mechanism is given by electron micrographs of TM formations.     

Dicyclohexylcarbodiimide and vanadate sensitive ATPase of lung lamellar bodies

Lung surfactant is synthesized in lung epithelial type II cells and stored in the lamellar bodies prior to its secretion onto the alveolar surface. The lamellar bodies, like other secretory organelles, maintain an ATP-dependent pH gradient that is sensitive to inhibitors of H⁺-ATPase. This report shows that the ATPase activity of lamellar bodies is enriched in a fraction prepared from lamellar bodies that were disrupted after isolation. The apparent V_max for this enzyme was 150 nmol ATP hydrolyzed per min per mg protein and apparent K_m for ATP was approximately 50 μM. The enzyme activity was sensitive to N-ethylmaleimide (NEM), dicyclohexylcarbodiimide (DCCD) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) (all inhibitors of vacuolar-type H⁺-ATPase) and vanadate (inhibitor of phosphoenzyme-type ATPase). Besides, the activity could also be inhibited with diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and Ca²⁺. Two proteins (of approximately 45 kDa and 17 kDa) of this fraction showed acid-stable phosphorylation with ATP. The labeling of proteins with ATP (-γ-³²P) could be chased with unlabelled ATP, suggesting that phosphorylation and dephosphorylation of these proteins is associated with the ATPase activity. Our results on inhibition characteristics of the enzyme activity suggest that besides a vacuolar type H⁺-ATPase, the lamellar bodies also contain a phosphoenzyme type ATPase that is sensitive to inhibitors of vacuolar type H⁺-ATPase.     

A role for diacylglycerol in antibacterial autophagy

Antibacterial autophagy is understood to be a key cellular immune response to invading microbes. However, the mechanism(s) by which bacteria are selected as targets of autophagy remain unclear. We recently identified diacylglycerol as a novel signaling molecule that targets bacteria to the autophagy pathway, and show that it acts via protein kinase C activation. We also found that Pkc1 is required for autophagy in yeast, indicating that this kinase plays a conserved role in autophagy regulation.Key words: bacteria, Salmonella, innate immunity, adaptor, lipid second messenger, diacylglycerol, ubiquitin, NDP52, p62, SQSTM1The mechanism by which bacteria and other subcellular targets are identified and degraded by the autophagy pathway is an area of intense research. Ubiquitin has been recently found to act as an essential signal required for the autophagy of bacteria and proteins. We have previously observed ubiquitin on autophagy-targeted Salmonella enterica serovar Typhimurium (S. Typhimurium) but were surprised to see that only 50% of these bacteria were positive for ubiquitin. This indicated the possibility that an alternate signal was required for efficient autophagic targeting of the nonubiquitinated population of these bacteria.We initially performed a screen quantifying the colocalization of different lipid second messengers (diacylglycerol (DAG), PtdIns(3)P, PtdIns(4,5)P₂, PtdIns(3,4) P₂, and PtdIns(3,4,5)P₃) with autophagytargeted (i.e., LC3⁺) S. Typhimurium. We observed that DAG preferentially localizes with LC3⁺ bacteria. A kinetic analysis revealed that maximal DAG colocalization with bacteria (45 min post-infection) precedes maximal autophagy of the bacteria (60 min post-infection). Using pharmacological agents, siRNA and dominant negative constructs we were able to determine that DAG localization to the bacteria requires the action of phospholipase D (PLD; phosphatidylcholine to phosphatidic acid conversion) and phosphatidic acid phosphatase (PAP; phosphatidic acid to DAG conversion). We observed that inhibition of these pathways significantly reduces DAG localization to bacteria as well as concomitant autophagy of the bacteria, indicating a role for this lipid second messenger in the regulation of this process.Having determined that DAG is necessary for autophagy of bacteria we subsequently wanted to identify the effector through which it was signaling. Conventional and novel isoforms of the protein kinase C (PKC) family contain DAG-binding C1 domains. Accordingly, we targeted PKC isoforms using pharmacological agents, siRNA and knockout cell lines and were able to determine that DAG is signaling through the δ isoform of PKC. Inhibition of this serine/threonine kinase results in significant inhibition of antibacterial autophagy. Furthermore, bacterial replication in PKCδ knockout mouse embryonic fibroblasts is significantly higher compared to control fibroblasts, consistent with previous observations demonstrating that autophagy impairs intracellular replication of S. Typhimurium (Birmingham et al. 2006).We addressed the possibility that DAG and ubiquitin are functioning in a cooperative manner to target Salmonella for degradation by autophagy. We simultaneously inhibited both pathways using siRNA or pharmacological agents and observed additive inhibitory effects on autophagy of the bacteria. While this is indicative of two independent pathways, we cannot discount the possibility that there is still cooperation between the two pathways, especially as we did observe a small population of bacteria that were positive for both DAG and ubiquitin (Fig. 1). There are also a number of technical limitations in the methods we used, such as detection levels of the probes and antibodies that warrant caution in concluding that the two pathways are completely independent. Nonetheless, our studies clearly demonstrate a role for both DAG (Shahnazari et al. 2010) and ubiquitin (Zheng et al. 2009) in autophagy of S. Typhimurium. Future studies are required to further examine how these signals contribute to regulation of antibacterial autophagy.Open in a separate windowFigure 1Autophagic targeting of Salmonella Typhimurium. Invading S. Typhimurium can be targeted to the autophagy pathway by two independent signaling mechanisms. The first requires ubiquitin and the autophagy adaptors p62 and NDP52. The second requires DAG generation and PKCδ function. DAG generation on the SCV may occur through interaction of the SCV with DAG-positive endocytic vesicles (pathway 1) or through direct DAG production on the SCV (pathway 2). SCV, Salmonella-containing vacuole; PA, phosphatidic acid; DAG, diacylglycerol; PAP, phosphatidic acid phosphatase; PKCδ, protein kinase C delta; Ub, ubiquitin.Having characterized this pathway in antibacterial autophagy we were interested in determining whether these components were required for general autophagy. We therefore tested whether DAG localizes with rapamycin-induced autophagosomes. We observed DAG on these compartments and also found a requirement for PAP and PKCδ in this process. Other PKC isoforms are involved in alternate types of autophagy including ER stress-induced autophagy (Sakaki et al. 2008) as well as hypoxia-induced autophagy (Chen et al. 2009). As a result, we were interested in determining whether PKC function in autophagy was evolutionarily conserved. We therefore tested a role for the yeast ortholog, Pkc1, in this process and observed that it is required for starvation-induced autophagy in Saccharomyces cerevisiae.Having identified and characterized a novel signal and effector for antibacterial autophagy, further work still remains to be done in order to obtain a complete picture of this process. This includes additional study of the mechanism by which DAG is generated and the subcellular localization of PLD and PAP during this process. It is possible that DAG⁺ endocytic vesicles fuse with the Salmonella-containing vacuole (SCV) coating this compartment with DAG (pathway 1, see Fig. 1). It is also possible that both PLD and PAP function directly on the SCV, converting phosphatidylcholine to DAG via the phosphatidic acid intermediate (pathway 2, Fig. 1).More work also needs to be done to dissect DAG and ubiquitin signaling contributions to this pathway. Questions to be answered include the identification of the ubiquitinated protein(s) on the SCV, which may be host or bacterial proteins. Additionally, while we know that DAG is present on the SCV we do not yet know the signal that induces its generation. One intriguing possibility is that DAG generation occurs in response to bacterial-induced damage to the SCV during invasion. To date, PKC has been implicated in at least three different types of autophagy, and the possibility exists that other PKC isoforms (DAG responsive or not) are also involved in this process.     

Dicyclohexylcarbodiimide and vanadate sensitive ATPase of lung lamellar bodies.

Lung surfactant is synthesized in lung epithelial type II cells and stored in the lamellar bodies prior to its secretion onto the alveolar surface. The lamellar bodies, like other secretory organelles, maintain an ATP-dependent pH gradient that is sensitive to inhibitors of H(+)-ATPase. This report shows that the ATPase activity of lamellar bodies is enriched in a fraction prepared from lamellar bodies that were disrupted after isolation. The apparent Vmax for this enzyme was 150 nmol ATP hydrolyzed per min per mg protein and apparent Km for ATP was approximately 50 microM. The enzyme activity was sensitive to N-ethylmaleimide (NEM), dicyclohexylcarbodiimide (DCCD) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) (all inhibitors of vacuolar-type H(+)-ATPase) and vanadate (inhibitor of phosphoenzyme-type ATPase). Besides, the activity could also be inhibited with diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and Ca2+. Two proteins (of approximately 45 kDa and 17 kDa) of this fraction showed acid-stable phosphorylation with ATP. The labeling of proteins with ATP (-gamma-32P) could be chased with unlabelled ATP, suggesting that phosphorylation and dephosphorylation of these proteins is associated with the ATPase activity. Our results on inhibition characteristics of the enzyme activity suggest that besides a vacuolar type H(+)-ATPase, the lamellar bodies also contain a phosphoenzyme type ATPase that is sensitive to inhibitors of vacuolar type H(+)-ATPase.     

Comparative proteomic analysis of lung lamellar bodies and lysosome-related organelles

Pulmonary surfactant is a complex mixture of lipids and proteins that is essential for postnatal function. Surfactant is synthesized in alveolar type II cells and stored as multi-bilayer membranes in a specialized secretory lysosome-related organelle (LRO), known as the lamellar body (LB), prior to secretion into the alveolar airspaces. Few LB proteins have been identified and the mechanisms regulating formation and trafficking of this organelle are poorly understood. Lamellar bodies were isolated from rat lungs, separated into limiting membrane and core populations, fractionated by SDS-PAGE and proteins identified by nanoLC-tandem mass spectrometry. In total 562 proteins were identified, significantly extending a previous study that identified 44 proteins in rat lung LB. The lung LB proteome reflects the dynamic interaction of this organelle with the biosynthetic, secretory and endocytic pathways of the type II epithelial cell. Comparison with other LRO proteomes indicated that 60% of LB proteins were detected in one or more of 8 other proteomes, confirming classification of the LB as a LRO. Remarkably the LB shared 37.8% of its proteins with the melanosome but only 9.9% with lamellar bodies from the skin. Of the 229 proteins not detected in other LRO proteomes, a subset of 34 proteins was enriched in lung relative to other tissues. Proteins with lipid-related functions comprised a significant proportion of the LB unique subset, consistent with the major function of this organelle in the organization, storage and secretion of surfactant lipid. The lung LB proteome will facilitate identification of molecular pathways involved in LB biogenesis, surfactant homeostasis and disease pathogenesis.     

Role of lamellar bodies in the biosynthesis of phosphatidylcholine in mouse lung.

1. A lamellar body-enriched fraction was isolated from whole lung homogenates of mouse lung and its contamination with microsomes, mitochondria, and cytosol protein assessed by marker enzyme analyses. 2. By measuring the activity of cholinephosphotransferase (EC 2.7.8.2) in varying amounts of microsomes in the presence and absence of a fixed quantity of lamellar bodies, it could be demonstrated unequivocally that lamellar bodies of mouse lung lack the capacity to synthesize phosphatidylcholine de novo. 3. A similar approach allowed the conclusion that lamellar bodies of mouse lung do not contain lysophosphatidylcholine acyltransferase (EC 2.3.1.23) and lysophosphatidylcholine:lysophosphatidylcholine acyltransferase (EC 2.3.1.--), enzymes which play a putative role in the formation of pulmonary 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine. The activities of these enzymes observed in lamellar body fractions could be attributed completely to contaminating microsomes and cytosol respectively. 4. Lamellar bodies contributed to the activity of microsomal lysophosphatidylcholine acyltransferase by a cooperative effect. The possible role of this cooperation in the biosynthesis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine is discussed.     

A role for diacylglycerol in antibacterial autophagy

Antibacterial autophagy is understood to be a key cellular immune response to invading microbes. However, the mechanism(s) by which bacteria are selected as targets of autophagy remain unclear. We recently identified diacylglycerol as a novel signaling molecule that targets bacteria to the autophagy pathway, and show that it acts via protein kinase C activation. We also found that Pkc1 is required for autophagy in yeast, indicating that this kinase plays a conserved role in autophagy regulation.     

Secretagogues of lung surfactant increase annexin A7 localization with ABCA3 in alveolar type II cells

Membrane fusion between the lamellar bodies and plasma membrane is an obligatory event in the secretion of lung surfactant. Previous studies have postulated a role for annexin A7 (A7) in membrane fusion during exocytosis in some cells including alveolar type II cells. However, the intracellular trafficking of A7 during such fusion is not described. In this study, we investigated association of endogenous A7 with lamellar bodies in alveolar type II cells following treatment with several secretagogues of lung surfactant. Biochemical studies with specific antibodies showed increased membrane-association of cell A7 in type II cells stimulated with agents that increase secretion through different signaling mechanisms. Immuno-fluorescence studies showed increased co-localization of A7 with ABCA3, the lamellar body marker protein. Because these agents increase surfactant secretion through activation of PKC and PKA, we also investigated the effects of PKC and PKA inhibitors, bisindolylmaleimideI (BisI) and H89, respectively, on A7 partitioning. Western blot analysis showed that these inhibitors prevented secretagogue-mediated A7 increase in the membrane fractions. These inhibitors also blocked increased co-localization of A7 with ABCA3 in secretagogue-treated cells, as revealed by immuno-fluorescence studies. In vitro studies with recombinant A7 showed phosphorylation with PKC and PKA. The cell A7 was also phosphorylated in cells treated with surfactant secretagogues. Thus, our studies demonstrate that annexin A7 relocates to lamellar bodies in a phosphorylation-dependent manner. We suggest that activation of protein kinase promotes phosphorylation and membrane-association of A7 presumably to facilitate membrane fusion during lung surfactant secretion.     

Giant lamellar bodies in pulmonary MALT lymphoma. A case report

BACKGROUND: Giant lamellar bodies are laminated, scroll-like whorls seen within alveolar spaces and have been occasionally observed in sclerosing hemangioma of the lung. However, to the best of our knowledge, the cytologic findings of giant lamellar bodies have not been reported. We describe cytologic findings of giant lamellar bodies associated with pulmonary mucosa-associated lymphoid tissue (MALT) lymphoma. CASE: A 72-year-old male had a pulmonary mass measuring 2.0 x 1.4 x 1.5 cm. Cytologic smears imprinted from a cut surface of the resected mass revealed a large number of concentrically laminated structures, giant lamellar bodies, measuring 15-40 microns in diameter. Necrotic cellular remnants were occasionally observed in the center of the structures. In the background, small to medium-sized lymphoid cells and plasmacytoid cells were observed. Histologic diagnosis of the tumor was IgG, kappa type, MALT lymphoma. An aggregate of giant lamellar bodies was observed within entrapped, dilated alveolar spaces lined with hypertrophied, type II pneumocytes. Immunohistochemically, the giant lamellar bodies were positive for KL-6. CONCLUSION: Giant lamellar bodies may be derived from surfactant and necrotic type II pneumocytes and may be observed cytologically in cases of pulmonary MALT lymphoma.     

A role for lipid in myoblast fusion

Alteration of the membrane fatty acyl composition modulates the fusion of myoblasts into multinucleate myotubes. The rate of fusion after addition of calcium to 50–52 hour cultures of chick pectoral myoblasts is markedly inhibited in cells possessing acyl chains enriched in elaidate and is enhanced in those enriched in oleate. The modulations appear to occur after the cells have recognized one another and adhered strongly but before the membranes have united. These observations lead to a hypothesis for membrane union (fusion) in which the lipids participate directly perhaps by a mechanism analogous to that proposed for the fusion of lipid vesicles.     

Phospholipid composition of lamellar bodies formed by fetal rabbit lung type II cells in organ culture

Lung tissue obtained from fetal rabbits of 23 days gestational age was maintained in organ culture to study the in vitro formation of lamellar body phospholipids. During the culture period, the epithelium of the prealveolar ducts of the explants differentiated to form type II pneumonocytes. After 8 days in culture, the explants were harvested, homogenized, and two lamellar body fractions were isolated by sucrose density gradient centrifugation. The lamellar body fraction which best retained the distinct multilamellar structure was recovered at the interface between a solution of buffer without sucrose and buffer containing 0.41 m sucrose. The phospholipid compositions of both lamellar body fractions were similar to those reported for lamellar bodies and surfactant isolated from fetal rabbit lung, with the exception of a slightly higher phosphatidylethanolamine content. The disaturated phosphatidylcholine content of the lamellar body fractions, expressed as a percentage of total lipid phosphorus, was not influenced by the presence of palmitate in the medium.     

Evidence for a high activity of sphingomyelin biosynthesis by phosphocholine transfer from phosphatidylcholine to ceramides in lung lamellar bodies

Biosynthesis of sphingomyelin from ceramides was investigated in lung subcellular fractions by incubating a lyophilized mixture of albumin and subcellular fraction (0.1-0.2 mg of protein) coated with [acyl-14C]-ceramide and phosphatidyl[methyl-3H]choline in Tris-buffer. The lamellar-body-rich fraction exhibited the highest specific activity for sphingomyelin biosynthesis measured by 14C incorporation into sphingomyelins or by [3H]phosphocholine transfer from phosphatidylcholines. Plasma membranes formed the next most active fraction, followed by the 'smooth' and, then, the 'rough' endoplasmic reticulum. Sphingomyelin biosynthesis by lamellar bodies was optimum at pH 7.4 and was inhibited by sphingomyelins formed. Slight inhibitory effects were also observed with Mn2+, Ca2+ and lysophosphatidylcholine. Phosphocholine transfer from CDPcholine was not observed under the reaction conditions employed. Ceramide conversion and phosphocholine transfer increased with ceramide concentration to reach a maximum at about 0.06 mM. The highest conversion rate was observed when 18:1 ceramide was used as an acceptor. When 1-palmitoyl-2-oleoylphosphatidylcholine was the phosphocholine donor, the overall biosynthesis of sphingomyelin was much higher than when using dipalmitoylphosphatidylcholine. These results suggest the possible involvement of the studied reaction in the control of the degree of saturation of the surfactant phosphatidylcholine.