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Abstract Background The pulmonary surfactant that lines the air–liquid surface within alveoli is a protein–lipid mixture essential for gas exchange. Surfactant lipids and proteins are synthesized and stored in the lamellar body (LB) before being secreted from alveolar type II (AT2) cells. The molecular and cellular mechanisms that regulate these processes are incompletely understood. We previously identified an essential role of general control of amino acid synthesis 5 like 1 (GCN5L1) and the biogenesis of lysosome-related organelle complex 1 subunit 1 (BLOS1) in surfactant system development in zebrafish. Here, we explored the role of GCN5L1 in pulmonary surfactant regulation. Method GCN5L1 knockout cell lines were generated with the CRISPR/Cas9 system. Cell viability was analyzed by MTT assay. Released surfactant proteins were measured by ELISA. Released surfactant lipids were measured based on coupled enzymatic reactions. Gene overexpression was mediated through lentivirus. The RNA levels were detected through RNA-sequencing (RNA-seq) and quantitative reverse transcription (qRT)- polymerase chain reaction (PCR). The protein levels were detected through western blotting. The cellular localization was analyzed by immunofluorescence. Morphology of the lamellar body was analyzed through transmission electron microscopy (TEM), Lysotracker staining, and BODIPY phosphatidylcholine labeling. Results Knocking out GCN5L1 in MLE-12 significantly decreased the release of surfactant proteins and lipids. We detected the downregulation of some surfactant-related genes and misregulation of the ROS–Erk–Foxo1–Cebpα axis in mutant cells. Modulating the activity of the axis or reconstructing the mitochondrial expression of GCN5L1 could partially restore the expression of these surfactant-related genes. We further showed that MLE-12 cells contained many LB-like organelles that were lipid enriched and positive for multiple LB markers. These organelles were smaller in size and accumulated in the absence of GCN5L1, indicating both biogenesis and trafficking defects. Accumulated endogenous surfactant protein (SP)-B or exogenously expressed SP-B/SP-C in adenosine triphosphate-binding cassette transporterA3 (ABCA3)-positive organelles was detected in mutant cells. GCN5L1 localized to the mitochondria and LBs. Reconstruction of mitochondrial GCN5L1 expression rescued the organelle morphology but failed to restore the trafficking defect and surfactant release, indicating specific roles associated with different subcellular localizations. Conclusions In summary, our study identified GCN5L1 as a new regulator of pulmonary surfactant that plays a role in the biogenesis and positioning/trafficking of surfactant-containing LBs.

Background: The pulmonary surfactant that lines the air–liquid surface within alveoli is a protein–lipid mixture essential for gas exchange. Surfactant lipids and proteins are synthesized and stored in the lamellar body (LB) before being secreted from alveolar type II (AT2) cells. The molecular and cellular mechanisms that regulate these processes are incompletely understood. We previously identified an essential role of general control of amino acid synthesis 5 like 1 (GCN5L1) and the biogenesis of lysosome-related organelle complex 1 subunit 1 (BLOS1) in surfactant system development in zebrafish. Here, we explored the role of GCN5L1 in pulmonary surfactant regulation. Method: GCN5L1 knockout cell lines were generated with the CRISPR/Cas9 system. Cell viability was analyzed by MTT assay. Released surfactant proteins were measured by ELISA. Released surfactant lipids were measured based on coupled enzymatic reactions. Gene overexpression was mediated through lentivirus. The RNA levels were detected through RNA-sequencing (RNA-seq) and quantitative reverse transcription (qRT)- polymerase chain reaction (PCR). The protein levels were detected through western blotting. The cellular localization was analyzed by immunofluorescence. Morphology of the lamellar body was analyzed through transmission electron microscopy (TEM), Lysotracker staining, and BODIPY phosphatidylcholine labeling. Results: Knocking out GCN5L1 in MLE-12 significantly decreased the release of surfactant proteins and lipids. We detected the downregulation of some surfactant-related genes and misregulation of the ROS–Erk–Foxo1–Cebpα axis in mutant cells. Modulating the activity of the axis or reconstructing the mitochondrial expression of GCN5L1 could partially restore the expression of these surfactant-related genes. We further showed that MLE-12 cells contained many LB-like organelles that were lipid enriched and positive for multiple LB markers. These organelles were smaller in size and accumulated in the absence of GCN5L1, indicating both biogenesis and trafficking defects. Accumulated endogenous surfactant protein (SP)-B or exogenously expressed SP-B/SP-C in adenosine triphosphate-binding cassette transporterA3 (ABCA3)-positive organelles was detected in mutant cells. GCN5L1 localized to the mitochondria and LBs. Reconstruction of mitochondrial GCN5L1 expression rescued the organelle morphology but failed to restore the trafficking defect and surfactant release, indicating specific roles associated with different subcellular localizations. Conclusions: In summary, our study identified GCN5L1 as a new regulator of pulmonary surfactant that plays a role in the biogenesis and positioning/trafficking of surfactant-containing LBs. [ABSTRACT FROM AUTHOR]

Atopic dermatitis (AD), the most common inflammatory skin disorder, is a multifactorial disease characterized by a genetic predisposition, epidermal barrier disruption, a strong T helper (Th) type 2 immune reaction to environmental antigens and an altered cutaneous microbiome. Microbial dysbiosis characterized by the prevalence of Staphylococcus aureus (S. aureus) has been shown to exacerbate AD. In recent years, in vitro models of AD have been developed, but none of them reproduce all of the pathophysiological features. To better mimic AD, we developed reconstructed human epidermis (RHE) exposed to a Th2 pro-inflammatory cytokine cocktail and S. aureus. This model well reproduced some of the vicious loops involved in AD, with alterations at the physical, microbial and immune levels. Our results strongly suggest that S. aureus acquired a higher virulence potential when the epidermis was challenged with inflammatory cytokines, thus later contributing to the chronic inflammatory status. Furthermore, a topical application of a Castanea sativa extract was shown to prevent the apparition of the AD-like phenotype. It increased filaggrin, claudin-1 and loricrin expressions and controlled S. aureus by impairing its biofilm formation, enzymatic activities and inflammatory potential. [ABSTRACT FROM AUTHOR]

Background: Lamellar bodies are produced by pneumocyte type II cells in the lung alveoli. Lamellar bodies are present in amniotic fluid in increasing quantities as gestation advances, 1 – 5 µm in size, similar in size to small platelets and can be counted on most electronic cell counters in hematology analyzer. Lamellar body count is useful for prediction of fetal lung maturity and neonatal respiratory distress syndrome. The current gold standard for determination of fetal lung maturity is the evaluation of phospholipids in amniotic fluid samples by thin-layer chromatography, but it is time-consuming and not continuously available at most institutions. In this study we compare Cell Dyn Emerald and Cell Dyn Ruby method, which is expected to be a review for lamellar body count method. Purpose: The aim of this study was to analyze lamellar body count with Cell Dyn Emerald and Cell Dyn Ruby method on preterm birth. Methods : This was a cross sectional study. Thirty three samples study were inpatient’s amniotic fluid with premature rupture of membranes in Obstetry and Gynecology ward emergency room Dr. Soetomo Hospital Surabaya. Lamellar body count was counted with Cell Dyn emerald and Cell Dyn Ruby method. The statistical differences were assessed using the ANOVA test . Results : The results showed significant differences (t=49,04), lamellar body count with Cell Dyn Ruby method was much lower than Cell Dyn Emerald method. The lowest result with Cell Dyn Ruby method was 3.38 x 103/µL and 17 x 103/ µL with Cell Dyn Emerald method. The highest results with Cell Dyn Ruby method was 98,2 x 103/ µL and 221 x 103/ µL with Cell Dyn Emerald method. Conclusion : Lamellar body count with impedance method (Cell Dyn Emerald) is significantly higher than optic method (Cell Dyn Ruby).

Lysosome‐related organelles (LROs) comprise a diverse group of cell type‐specific, membrane‐bound subcellular organelles that derive at least in part from the endolysosomal system but that have unique contents, morphologies and functions to support specific physiological roles. They include: melanosomes that provide pigment to our eyes and skin; alpha and dense granules in platelets, and lytic granules in cytotoxic T cells and natural killer cells, which release effectors to regulate hemostasis and immunity; and distinct classes of lamellar bodies in lung epithelial cells and keratinocytes that support lung plasticity and skin lubrication. The formation, maturation and/or secretion of subsets of LROs are dysfunctional or entirely absent in a number of hereditary syndromic disorders, including in particular the Hermansky‐Pudlak syndromes. This review provides a comprehensive overview of LROs in humans and model organisms and presents our current understanding of how the products of genes that are defective in heritable diseases impact their formation, motility and ultimate secretion. [ABSTRACT FROM AUTHOR]

Background The morbidity and mortality of hyaline membrane dis- ease (HMD) are quite high due to delayed diagnosis and intervention. Commonly, HMD occurs in preterm infants with surfactant deficiency because of lung immaturity. Lung maturity test could be performed using biochemical, biophysical, and amniotic fluid turbidity test. Objective To find out HMD prevalence and the value of shake test and lamellar body concentration in diagnosing HMD in preterm infants. Methods This was a cross-sectional study carried out at Hasan Sadikin Hospital Bandung on preterm infants born during October- December 2001. The shake test was performed using gastric fluid and amniotic fluid while the lamellar body concentration was per- formed using amniotic fluid. Results During the 3-month period, 571 infants were born, of 64 (11.2%) preterm infants, only 41 (64%) fulfilled the inclusion crite- ria; among those preterm infants, 14 (34%) suffered from respira- tory distress and 7 suffered from HMD (prevalence 17%). All HMD cases occurred in infants less than 32 weeks for gestational age. In 7 preterm infants with HMD, the shake test of gastric fluid ob- tained by lavage showed negative results in 3 and +1 in 4 infants; while the shake test of amniotic fluid revealed negative result in 5 and +1 in 2 infants. Lamellar body concentration of amniotic fluid was ≤18,000/ml in all HMD infants. Among three infants less than 32 weeks for gestational age who did not suffer from HMD, +1 shake test of gastric fluid was found in 2 infants and +2 in 1 infant; while shake test of amniotic fluid showed negative result in 1 infant and +1 in 2; the lamellar body concentration of amniotic fluid was ≤18,000/ml in 2 infants and >18,000/ml in 1 infant. Conclusions We concluded that HMD occurred in 17% of preterm infants. The shake test of gastric and amniotic fluids revealed nega- tive or +1 results whereas lamellar body concentration had value of less than or equal to 18,000/mL. More extensive studies are warranted to assess the validity (sensitivity, specificity and predic- tive values) of these measurements

In alveolar type II (AT II) cells, pulmonary surfactant (PS) is synthetized, stored and exocytosed from lamellar bodies (LBs), specialized large secretory organelles. By applying polarization microscopy (PM), we confirm a specific optical anisotropy of LBs, which indicates a liquid-crystalline mesophase of the stored surfactant phospholipids (PL) and an unusual case of a radiation-symmetric, spherocrystalline organelle. Evidence is shown that the degree of anisotropy is dependent on the amount of lipid layers and their degree of hydration, but unaffected by acutely modulating vital cell parameters like intravesicular pH or cellular energy supply. In contrast, physiological factors that perturb this structure include osmotic cell volume changes and LB exocytosis. In addition, we found two pharmaceuticals, Amiodarone and Ambroxol, both of which severely affect the liquid-crystalline order. Our study shows that PM is an easy, very sensitive, but foremost non-invasive and label-free method able to collect important structural information of PS assembly in live AT II cells which otherwise would be accessible by destructive or labor intense techniques only. This may open new approaches to dynamically investigate LB biosynthesis - the incorporation, folding and packing of lipid membranes - or the initiation of pathological states that manifest in altered LB structures. Due to the observed drug effects, we further suggest that PM provides an appropriate way to study unspecific drug interactions with alveolar cells and even drug-membrane interactions in general. [ABSTRACT FROM AUTHOR]

ABCG1, a member of the ATP binding cassette superfamily, facilitates the efflux of cholesterol from cells to HDL. In this study, we demonstrate that ABCG1 is expressed in cultured human keratinocytes and murine epidermis, and induced during keratinocyte differentiation, with increased levels in the outer epidermis. ABCG1 is regulated by liver X receptor (LXR) and peroxisome proliferator-activated receptor-δ (PPAR-δ) activators, cellular sterol levels, and acute barrier disruption. Both LXR and PPAR-δ activators markedly stimulate ABCG1 expression in a dose- and time-dependent fashion. PPAR-γ activators also increase ABCG1 expression, but to a lesser degree. In contrast, activators of PPAR-α, retinoic acid receptor, retinoid X receptor, and vitamin D receptor do not alter ABCG1 expression. In response to increased intracellular sterol levels, ABCG1 expression increases, whereas inhibition of cholesterol biosynthesis decreases ABCG1 expression. In vivo, ABCG1 is stimulated 3–6 h after acute barrier disruption by either tape stripping or acetone treatment, an increase that can be inhibited by occlusion, suggesting a potential role of ABCG1 in permeability barrier homeostasis. Although Abcg1-null mice display normal epidermal permeability barrier function and gross morphology, abnormal lamellar body (LB) contents and secretion leading to impaired lamellar bilayer formation could be demonstrated by electron microscopy, indicating a potential role of ABCG1 in normal LB formation and secretion.

The permeability barrier is required for terrestrial life and is localized to the stratum corneum, where extracellular lipid membranes inhibit water movement. The lipids that constitute the extracellular matrix have a unique composition and are 50% ceramides, 25% cholesterol, and 15% free fatty acids. Essential fatty acid deficiency results in abnormalities in stratum corneum structure function. The lipids are delivered to the extracellular space by the secretion of lamellar bodies, which contain phospholipids, glucosylceramides, sphingomyelin, cholesterol, and enzymes. In the extracellular space, the lamellar body lipids are metabolized by enzymes to the lipids that form the lamellar membranes. The lipids contained in the lamellar bodies are derived from both epidermal lipid synthesis and extracutaneous sources. Inhibition of cholesterol, fatty acid, ceramide, or glucosylceramide synthesis adversely affects lamellar body formation, thereby impairing barrier homeostasis. Studies have further shown that the elongation and desaturation of fatty acids is also required for barrier homeostasis. The mechanisms that mediate the uptake of extracutaneous lipids by the epidermis are unknown, but keratinocytes express LDL and scavenger receptor class B type 1, fatty acid transport proteins, and CD36. Topical application of physiologic lipids can improve permeability barrier homeostasis and has been useful in the treatment of cutaneous disorders.

The highly branched mammalian lung relies on surfactant, a mixture of phospholipids, cholesterol, and hydrophobic proteins, to reduce intraalveolar surface tension and prevent lung collapse. Human mutations in the ABCA3 transporter have been associated with childhood respiratory disease of variable severity and onset. Here, we report the generation of Abca3 null mice, which became lethargic and cyanotic and died within 1 h of birth. Tissue blots found ABCA3 expression was highest in lung but was also detectable in other tissues, including the kidney. Gross development of kidney and lung was normal in neonatal Abca3−/− pups, but the mice failed to inflate their lungs, leading to death from atelectatic respiratory failure. Ultrastructural analysis of the Abca3−/− lungs revealed an absence of surfactant from the alveolar space and a profound loss of mature lamellar bodies, the intracellular storage organelle for surfactant. Mass spectrometry measurement of >300 phospholipids in lung tissue taken from Abca3−/− mice showed a dramatic reduction of phosphatidylglycerol (PG) levels as well as selective reductions in phosphatidylcholine species containing short acyl chains. These results establish a requirement of ABCA3 for lamellar body formation and pulmonary surfactant secretion and suggest a unique and critical role for the transporter in the metabolism of pulmonary PG. They also demonstrate the utility of the Abca3 null mouse as a model for a devastating human disease.

The molecular basis involving adsorption of pulmonary surfactant at the respiratory air–liquid interface and the specific roles of the surfactant proteins SP-B and SP-C in this process have not been completely resolved. The reasons might be found in the largely unknown structural assembly in which surfactant lipids and proteins are released from alveolar type II cells, and the difficulties to sample, manipulate and visualize the adsorption of these micron-sized particles at an air–liquid interface under appropriate physiological conditions. Here, we introduce several approaches to overcome these problems. First, by immunofluorescence we could demonstrate the presence of SP-B and SP-C on the surface of exocytosed surfactant particles. Second, by sampling the released particles and probing their adsorptive capacity we could demonstrate a remarkably high rate of interfacial adsorption, whose rate and extent was dramatically affected by treatment with antibodies against SP-B and SP-C. The effect of both antibodies was additive and specific. Third, direct microscopy of an inverted air–liquid interface revealed that the blocking effect is due to a stabilization of the released particles when contacting the air–liquid interface, precluding their transformation and the formation of surface films. We conclude that SP-B and SP-C are acting as essential, preformed molecular keys in the initial stages of surfactant unpacking and surface film formation. We further propose that surfactant activation might be transduced by a conformational change of the surfactant proteins upon contact with surface forces acting on the air–liquid interface. [ABSTRACT FROM AUTHOR]

Phospholipids are a major class of lipids in epidermis, where they serve as a source of free fatty acids that are important for the maintenance of epidermal permeability barrier function. The phospholipid biosynthetic enzyme, 1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT), catalyzes the acylation of lysophosphatidic acid to form phosphatidic acid, the major precursor of all glycerolipids. We identified an expression pattern of AGPAT isoforms that is unique to epidermis, with relatively high constitutive expression of mouse AGPAT (mAGPAT) 3, 4, and 5 but low constitutive expression of mAGPAT 1 and 2. Localization studies indicate that all five isoforms of AGPAT were expressed in all nucleated layers of epidermis. Furthermore, rat AGPAT 2 and 5 mRNAs increased in parallel with both an increase in enzyme activity and permeability barrier formation late in rat epidermal development. Moreover, after two methods of acute permeability barrier disruption, mAGPAT 1, 2, and 3 mRNA levels increased rapidly and were sustained for at least 24 h. In parallel with the increase in mRNA levels, an increase in AGPAT activity also occurred.Because upregulation of mAGPAT mRNAs after tape-stripping could be partially reversed by artificial barrier restoration by occlusion, these studies suggest that an increase in the expression of AGPATs is linked to barrier requirements.

In many secretory cells actin and myosin are specifically recruited to the surface of secretory granules following their fusion with the plasma membrane. Actomyosin-dependent compression of fused granules is essential to promote active extrusion of cargo. However, little is known about molecular mechanisms regulating actin coat formation and contraction. Here, we provide a detailed kinetic analysis of the molecules regulating actin coat contraction on fused lamellar bodies in primary alveolar type II cells. We demonstrate that ROCK1 and myosin light chain kinase 1 (MLCK1, also known as MYLK) translocate to fused lamellar bodies and activate myosin II on actin coats. However, myosin II activity is not sufficient for efficient actin coat contraction. In addition, cofilin-1 and α-actinin translocate to actin coats. ROCK1-dependent regulated actin depolymerisation by cofilin-1 in cooperation with actin crosslinking by α-actinin is essential for complete coat contraction. In summary, our data suggest a complementary role for regulated actin depolymerisation and crosslinking, and myosin II activity, to contract actin coats and drive secretion. [ABSTRACT FROM AUTHOR]

In recent years P2X receptors have attracted increasing attention as regulators of exocytosis and cellular secretion. In various cell types P2X receptors have been found to stimulate vesicle exocytosis directly via Ca2+ influx and elevation of the intracellular Ca2+ concentration. Recently, a new role for P2X4 receptors as regulators of secretion emerged. Exocytosis of lamellar bodies (LBs), large storage organelles for lung surfactant, results in a local, fusion-activated Ca2+ entry (FACE) in alveolar type II epithelial cells. FACE is mediated via P2X4 receptors that are located on the limiting membrane of LBs and inserted into the plasma membrane upon exocytosis of LBs. The localized Ca2+ influx at the site of vesicle fusion promotes fusion pore expansion and facilitates surfactant release. In addition, this inward-rectifying cation current across P2X4 receptors mediates fluid resorption from lung alveoli. It is hypothesized that the concomitant reduction in the alveolar lining fluid facilitates insertion of surfactant into the air-liquid interphase thereby activating it. These findings constitute a novel role for P2X4 receptors in regulating vesicle content secretion as modulators of the secretory output during the exocytic post-fusion phase.

Ca2+-dependent regulation of fusion pore dilation and closure is a key mechanism determining the output of cellular secretion. We have recently described 'fusion-activated' Ca2+ entry (FACE) following exocytosis of lamellar bodies in alveolar type II cells. FACE regulates fusion pore expansion and facilitates secretion. However, the mechanisms linking this locally restricted Ca2+ signal and fusion pore expansion were still elusive. Here, we demonstrate that synaptotagmin-7 (Syt7) is expressed on lamellar bodies and links FACE and fusion pore dilation. We directly assessed dynamic changes in fusion pore diameters by analysing diffusion of fluorophores across fusion pores. Expressing wild-type Syt7 or a mutant Syt7 with impaired Ca2+-binding to the C2 domains revealed that binding of Ca2+ to the C2A domain facilitates FACE-induced pore dilation, probably by inhibiting translocation of complexin-2 to fused vesicles. However, the C2A domain hampered Ca2+- dependent exocytosis of lamellar bodies. These findings support the hypothesis that Syt7 modulates fusion pore expansion in large secretory organelles and extend our picture that lamellar bodies contain the necessary molecular inventory to facilitate secretion during the exocytic post-fusion phase. Moreover, regulating Syt7 levels on lamellar bodies appears to be essential in order that exocytosis is not impeded during the pre-fusion phase. [ABSTRACT FROM AUTHOR]

Macroautophagy is a catabolic process critical for the degradation of intracellular material, but its physiological functions in vertebrates are not fully understood. Here, we discuss our recent finding that macroautophagy plays a role in lamellar body maturation. The lamellar body is a lysosome-related organelle and stores phospholipid-containing surfactant complexes that reduce the surface tension of the air–water interface in order to inflate the airspace in lungs and swim bladders. In the epithelial cells of these organs, autophagosomes fuse with immature lamellar bodies to increase their size and lipid contents. This function is essential for respiration after birth in mice and for maintaining buoyancy in zebrafish. These findings unveil a novel function of macroautophagy in the maturation of surfactant-containing lamellar bodies. [ABSTRACT FROM AUTHOR]

The skin serves the vital function of providing a barrier between the hostile external environment and the host. While the skin has many important barrier functions, the two that are absolutely essential for survival are the barrier to the movement of water and electrolytes (permeability barrier) and the barrier against invasive and toxic microorganisms (antimicrobial barrier). Lipids play an essential role in the formation and maintenance of both the permeability and antimicrobial barriers. A hydrophobic extracellular lipid matrix in the stratum corneum composed primarily of ceramides, cholesterol, and free fatty acids provides the barrier to the movement of water and electrolytes. A variety of lipids, such as fatty alcohols, monoglycerides, sphingolipids, phospholipids, and in particular free fatty acids, have antimicrobial activity and contribute to the antimicrobial barrier. In addition to these essential functions, we will also review the ability of skin surface cholesterol to reflect alterations in systemic lipid metabolism and the risk of atherosclerosis.

Abstract: Lung surfactant secretion involves lamellar body docking and fusion with the plasma membrane in alveolar type II cells. Annexin A7 (A7) is postulated to play a role in membrane fusion during exocytosis. Our recent studies demonstrated increased co-localization of A7 with ABCA3 in lamellar bodies in type II cells stimulated with established secretagogues of lung surfactant. In this study, we investigated in vivo and in vitro interactions of A7 with the t-SNARE protein, SNAP23. Immuno-fluorescence studies showed time-dependent increases in co-localization of A7 with SNAP23 in PMA- and in A23187-stimulated cells. PMA and A23187 also caused a time-dependent increase in co-localization of ABCA3 with SNAP23. The relocation of A7 to SNAP23 domains was inhibited in the presence of PKC inhibitor, similar to that previously reported for co-localization of A7 with ABCA3. The interaction of A7 and SNAP23 was confirmed by affinity binding and by in vitro interaction of recombinant A7 and SNAP23 proteins. The in vitro binding of recombinant A7 (rA7) to GST-SNAP23 fusion protein was calcium-dependent. Phosphorylation of rA7 with PKC increased its in vitro binding to SNAP23 suggesting that a similar mechanism may operate during A7 relocation to t-SNARE domains. Thus, our studies demonstrate that annexin A7 may function in co-ordination with SNARE proteins and that protein kinase activation may be required for annexin A7 trafficking to the interacting membranes (lamellar bodies and plasma membrane) to facilitate membrane fusion during surfactant secretion. [Copyright &y& Elsevier]

Secretion of vesicular contents by exocytosis is a fundamental cellular process. Increasing evidence suggests that post-fusion events play an important role in determining the composition and quantity of the secretory output. In particular, regulation of fusion pore dilation and closure is considered a key regulator of the post-fusion phase. However, depending on the nature of the cargo, additional mechanisms might be essential to facilitate effective release. We have recently described that in alveolar type II (ATII) cells, lamellar bodies (LBs), which are secretory vesicles that store lung surfactant, are coated with actin following fusion with the plasma membrane. Surfactant, a lipoprotein complex, does not readily diffuse out of fused LBs following opening and dilation of the fusion pore. Using fluorescence microscopy, atomic force microscopy and biochemical assays, we present evidence that actin coating and subsequent contraction of the actin coat is essential to facilitate surfactant secretion. Latrunculin B prevents actin coating of fused LBs and inhibits surfactant secretion almost completely. Simultaneous imaging of the vesicle membrane and the actin coat revealed that contraction of the actin coat compresses the vesicle following fusion. This leads to active extrusion of vesicle contents. Initial actin coating of fused vesicles is dependent on activation of Rho and formin-dependent actin nucleation. Actin coat contraction is facilitated by myosin II. In summary, our data suggest that fusion pore opening and dilation itself is not sufficient for release of bulky vesicle cargos and that active extrusion mechanisms are required. [ABSTRACT FROM AUTHOR]

Abstract: ABCA3 mutations cause fatal surfactant deficiency and interstitial lung disease. ABCA3 protein is a lipid transporter indispensible for surfactant biogenesis and storage in lamellar bodies (LB). The protein folds in endoplasmic reticulum and is glycosylated in Golgi en route to the membrane of mature LB and their precursor multivesicular bodies (MVB). In immunoblots, C-terminally labeled ABCA3 appears as two protein bands of 150 and 190kDa. Using N- and C-terminal protein tags and hindering ABCA3 processing we show that the 150kDa protein represents the mature ABCA3 whose N-terminus is cleaved by a cysteine protease inside MVB/LB. Structured summary: MINT-7996633: Calnexin (uniprotkb:P27824) and ABCA3 (uniprotkb:Q99758) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT-7996380,MINT-7996593,MINT-7996607: LAMP3 (uniprotkb:Q9UQV4) and ABCA3 (uniprotkb:Q99758) colocalize (MI:0403) by fluorescence microscopy (MI:0416) [ABSTRACT FROM AUTHOR]

Several Long-Evans rat substrains carrying the phenotype of oculo-cutaneous albinism and bleeding diathesis are a rat model of Hermansky-Pudlak syndrome (HPS). The mutation responsible for the phenotype (Ruby) was identified as a point mutation in the initiation codon of Rab38 small GTPase that regulates intracellular vesicle transport. As patients with HPS often develop life-limiting interstitial pneumonia accompanied by abnormal morphology of alveolar type II cells, we investigated lung surfactant system in Long-Evans Cinnamon rats, one strain of the Ruby rats. The lungs showed conspicuous morphology of type II cells containing markedly enlarged lamellar bodies. Surfactant phosphatidylcholine and surfactant protein B were increased in lung tissues and lamellar bodies but not in alveolar lumen. Expression levels of mRNA for surfactant proteins A, B, C, and D were not altered. Isolated type II cells showed aberrant secretory pattern of newly synthesized [3H]phosphatidylcholine, i.e., decreased basal secretion and remarkably amplified agonist-induced secretion. [3H]phosphatidylcholine synthesis and uptake by type II cells were not altered. Thus Rab38-deficient type II cells appear to carry abnormality in lung surfactant secretion but not in synthesis or uptake. These results suggest that aberrant lung surfactant secretion may be involved in the pathogenesis of interstitial pneumonia in HPS. [ABSTRACT FROM AUTHOR]

The ATP-binding cassette transporter ABCA3 mediates uptake of choline-phospholipids into intracellular vesicles and is essential for surfactant metabolism in lung alveolar type II cells. We have shown previously that ABCA3 mutations in fatal surfactant deficiency impair intracellular localization or ATP hydrolysis of ABCA3 protein. However, the mechanisms underlying the less severe phenotype of patients with ABCA3 mutation are unclear. In this study, we characterized ABCA3 mutant proteins identified in pediatric interstitial lung disease (pILD). E292V (intracellular loop 1), E690K (adjacent to Walker B motif in nucleotide binding domain 1), and T1114M (8th putative transmembrane segment) mutant proteins are localized mainly in intracellular vesicle membranes as wild-type protein. Lipid analysis and sucrose gradient fractionation revealed that the transport function of E292V mutant protein is moderately preserved, whereas those of E690K and T1114M mutant proteins are severely impaired. Vanadate-induced nucleotide trapping and photoaffinity labeling of wild-type and mutant proteins using 8-azido[32P]ATP revealed an aberrant catalytic cycle in these mutant proteins. These results demonstrate the importance of a functional catalytic cycle in lipid transport of ABCA3 and suggest a pathophysiological mechanism of pILD due to ABCA3 mutation. [ABSTRACT FROM AUTHOR]

Abstract: The pulmonary surfactant system constitutes an excellent example of how dynamic membrane polymorphism governs some biological functions through specific lipid–lipid, lipid–protein and protein–protein interactions assembled in highly differentiated cells. Lipid–protein surfactant complexes are assembled in alveolar pneumocytes in the form of tightly packed membranes, which are stored in specialized organelles called lamellar bodies (LB). Upon secretion of LBs, surfactant develops a membrane-based network that covers rapidly and efficiently the whole respiratory surface. This membrane-based surface layer is organized in a way that permits efficient gas exchange while optimizing the encounter of many different molecules and cells at the epithelial surface, in a cross-talk essential to keep the whole organism safe from potential pathogenic invaders. The present review summarizes what is known about the structure of the different forms of surfactant, with special emphasis on current models of the molecular organization of surfactant membrane components. The architecture and the behaviour shown by surfactant structures in vivo are interpreted, to some extent, from the interactions and the properties exhibited by different surfactant models as they have been studied in vitro, particularly addressing the possible role played by surfactant proteins. However, the limitations in structural complexity and biophysical performance of surfactant preparations reconstituted in vitro will be highlighted in particular, to allow for a proper evaluation of the significance of the experimental model systems used so far to study structure–function relationships in surfactant, and to define future challenges in the design and production of more efficient clinical surfactants. [Copyright &y& Elsevier]

Abstract: ABCA3 is proposed to function as a lung surfactant lipid transporter. Here we report ABCA3-dependent lipid uptake into intracellular vesicles in lung adenocarcinoma A549 cells. A549 cells stably expressing GFP-tagged wild-type ABCA3 (A549/ABCA3WT) had larger LAMP3-positive vesicles than their parental cells as well as A549 cells expressing a Walker A motif mutant (A549/ABCA3N568D). The choline-phospholipids level in A549/ABCA3WT was increased 1.25-fold compared to that in A549 and A549/ABCA3N568D cells, while the cholesterol levels were similar. Sucrose gradient fractionation analysis in A549/ABCA3WT cells revealed that choline-phospholipids were enriched in low-density and nile red-positive vesicles. Electronmicroscopic analysis showed multilamellar vesicles in A549/ABCA3WT cells. These results indicate that ABCA3 mediates ATP-dependent choline-phospholipids uptake into intracellular vesicles. [Copyright &y& Elsevier]

Abstract: Drug-induced phospholipidosis is characterized by intracellular accumulation of phospholipids with lamellar bodies, most likely from an impaired phospholipid metabolism of the lysosome. Organs affected by phospholipidosis exhibit inflammatory reactions and histopathological changes. Despite significant advances in the understanding of drug-altered lipid metabolism, the relationship between impaired phospholipid metabolism and drug-induced toxicity remains enigmatic. Here we review molecular features of inheritable lysosomal storage disorders as a molecular mimicry of drug-induced phospholipidosis for an improved understanding of adverse drug reaction. [Copyright &y& Elsevier]

Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disease caused by abnormalities in the synthesis and/or trafficking of lysosome-related organelles (LROs) including melanosomes, lamellar bodies of lung type II cells and platelet dense granules. At least 15 genes cause HPS in mice, with a significant number specifying novel subunits of protein complexes termed BLOCs ( Biogenesis of Lysosome-related Organelles Complexe s). To ascertain whether BLOC complexes functionally interact in vivo, mutant mice doubly or triply deficient in protein subunits of the various BLOC complexes and/or the AP-3 adaptor complex were constructed and tested for viability and for abnormalities of melanosomes, lung lamellar bodies and lysosomes. All mutants, including those deficient in all three BLOC complexes, were viable though the breeding efficiencies of multiple mutants involving AP-3 were severely compromised. Interactions of BLOC protein complexes with each other and with AP-3 to affect most LROs were apparent. However, these interactions were tissue and organelle dependent. These studies document novel biological interactions of BLOC and AP-3 complexes in the biosynthesis of LROs and assess the role(s) of HPS protein complexes in general health and physiology in mammals. Double and triple mutant HPS mice provide unique and practical experimental advantages in the study of LROs. [ABSTRACT FROM AUTHOR]

Most epidermal functions can be considered as protective, or more specifically, as defensive in nature. Yet, the term “barrier function” is often used synonymously with only one such defensive function, though arguably its most important, i.e., permeability barrier homeostasis. Regardless of their relative importance, these protective cutaneous functions largely reside in the stratum corneum (SC). In this review, I first explore the ways in which the multiple defensive functions of the SC are linked and interrelated, either by their shared localization or by common biochemical processes; how they are co-regulated in response to specific stressors; and how alterations in one defensive function impact other protective functions. Then, the structural and biochemical basis for these defensive functions is reviewed, including metabolic responses and signaling mechanisms of barrier homeostasis. Finally, the clinical consequences and therapeutic implications of this integrated perspective are provided. [ABSTRACT FROM AUTHOR]

Pulmonary surfactant is secreted by alveolar type II cells as lipid-rich, densely packed lamellar body-like particles (LBPs). The particulate nature of released LBPs might be the result of structural and/or thermodynamic forces. Thus mechanisms must exist that promote their transformation into functional units. To further define these mechanisms, we developed methods to follow LBPs from their release by cultured cells to insertion in an air-liquid interface. When released, LBPs underwent structural transformation, but did not disperse, and typically preserved a spherical appearance for days. Nevertheless, they were able to modify surface tension and exhibited high surface activity when measured with a capillary surfactometer. When LBPs inserted in an air-liquid interface were analyzed by fluorescence imaging microscopy, they showed remarkable structural transformations. These events were instantaneous but came to a halt when the interface was already occupied by previously transformed material or when surface tension was already low. These results suggest that the driving force for LBP transformation is determined by cohesive and tensile forces acting on these particles. They further suggest that transformation of LBPs is a self-regulated interfacial process that most likely does not require structural intermediates or enzymatic activation. [ABSTRACT FROM AUTHOR]

The lamellar body (LB), a concentric structure loaded with surfactant proteins and phospholipids, is an organelle specific to type 2 alveolar epithelial cells (AT2). However, the origin of LBs has not been fully elucidated. We have previously reported that autophagy regulates Weibel-Palade bodies (WPBs) formation, and here we demonstrated that autophagy is involved in LB maturation, another lysosome-related organelle. We found that during development, LBs were transformed from autophagic vacuoles containing cytoplasmic contents such as glycogen. Fusion between LBs and autophagosomes was observed in wild-type neonate mice. Moreover, the markers of autophagic activity, microtubule-associated protein 1 light chain 3B (LC3B), largely co-localized on the limiting membrane of the LB. Both autophagy-related gene 7 ( Atg7 ) global knockout and conditional Atg7 knockdown in AT2 cells in mice led to defects in LB maturation and surfactant protein B production. Additionally, changes in autophagic activity altered LB formation and surfactant protein B production. Taken together, these results suggest that autophagy plays a critical role in the regulation of LB formation during development and the maintenance of LB homeostasis during adulthood., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Recently, we established keratin 10-deficient mice, serving as a model for the hyperkeratotic skin disorder epidermolytic hyperkeratosis. The considerable ichthyosis in these mice suggested alterations in terminal differentiation and in the formation of a functional epidermal barrier. Here, we report on the ultrastructural organization and composition of the stratum corneum lipids and on the expression of two major cornified envelope proteins. Electron microscopy of ruthenium tetroxide postfixed skin samples demonstrated a normal extrusion and morphology of lamellar bodies as well as the formation of bona fide lamellar layers in neonatal keratin 10-deficient mice. When we studied the composition of the major stratum corneum lipids, however, we found significant changes. Most importantly, the analysis of ceramide subpopulations revealed that the total amount of ceramide 2 was elevated in keratin 10-deficient mice, whereas ceramides 1, 3, 4, and 5 were decreased among total stratum corneum lipids. The amount of the ceramide precursors sphingomyelin and glucosylceramide was reduced in the stratum corneum without accompanying changes in the mRNA coding for acid sphingomyelinase. Notably, we found an increased mRNA and protein content for involucrin in neonatal keratin 10-deficient mice, whereas the expression of loricrin was not changed. Our data demonstrate that, although the formation of lipid layers in the stratum corneum appeared to be normal, its lipid composition is significantly altered in keratin 10-deficient mice. [ABSTRACT FROM AUTHOR]

The epidermal cutaneous permeability barrier can be disrupted by treatment with topical solvents. Recent studies have shown that barrier recovery, measured by the recovery of transepidermal water loss towards normal, is inhibited by high extracellular Ca++ and K+, and accelerated by low extracellular concentrations of these ions. To examine the effects of Ca++ or K+ fluxes on barrier recovery, we tested the effects on transepidermal water loss recovery of agents that modify these fluxes. K+ channel agonists or blockers modified the inhibitory effects on barrier recovery induced by raised extracellular Ca++ and K+. In addition, Na+/K+ adenosine 5' triphosphatase inhibitors reversed the inhibitory effects of high extracellular Ca++ and K+. Our results suggest that barrier recovery requires both Ca++ and K+ fluxes and are consistent with the hypothesis that both verapamil or dihydropyridine-sensitive Ca++-permeable channels and Ca++-sensitive K+ channels participate in epidermal permeability barrier homeostasis. [ABSTRACT FROM AUTHOR]