Conversion of lamellar body membranes into tubular myelin in alveoli of fetal rat lungs

Fluid-filled lumina of fetal rat lungs contain lamellar bodies (LBs) as well as tubular myelin (TM), both of which are thought to be stores of phospholipid-rich pulmonary surfactant. The alveolar epithelium is believed to secrete LBs, but neither the origin nor the mechanism of TM formation is entirely certain. The main objective of this study was to determine the relationship between secreted LBs and TM and to define membrane phenomena which occur during TM formation. I examined lung tissues of 20-21 day-old fetuses (day 22 = term) using transmission and high voltage transmission electron microscopy and cytochemistry. My findings indicate that secreted LBs, identified by the presence of an acid-phosphatase reactive core, are the precursor of TM. Secreted LBs are highly organized structures which contain structurally specialized areas, one of which is a "mini-lattice" structure similar to TM. During TM formation, fuzzes or 8.0-nm diameter particles appear on transition membranes, although LB membranes appear to lack both structures. Similar particles are present on TM membranes and are generally associated with membrane intersections. My results provide evidence that TM is formed from LBs within the alveolar lumen by mechanisms which may be complex.     

Alveolar type II cells: studies on the mode of release of lamellar bodies.

There is increasing evidence that type II alveolar cells are capable of synthesizing surface active material like that obtained from the airways. However a number of problems remain to be solved before it can be stated conclusively that type II cells synthesize the surface active material of the terminal airspace. Among these problems is that of secretion. A number of previous studies have given evidence of the release of lamellar bodies by merocrine secretion. In this study morphologic evidence is presented which supports the view that secretion of lamellar bodies is accomplished by exocytosis. At the apical surface of type II cells, sites can be found where the limiting membrane of the lamellar body is clearly fused with the type II cell plasma membrane and an open channel exists between the contents of the lamellar body and the alveolar space. At these sites the lamellar contents extrude into the airspace with consequent loss of the highly compact organization of intracellular lamellar bodies. The intactness and continuity of the membranes can be traced for the full extent of the exocytosis site. Freeze-etch replicas of the membranes of type II cells show depressions which may represent the sites of discharged lamellae. In addition, tongue-like folds are seen which could be explained as the extensions of cytoplasm which surround the releasing lamellar body and which may flap over the exocytosis pit after discharge. Micrographs of the alveolar space show disorganized lamellar whorls which appear to be unravelling to produce tubular myelin. In view of the unusually large size and lipid composition of lamellar bodies, a mechanism involving hydration of mucopolysaccharide contents as an aid to expulsion of lamellar contents is suggested.     

Immunocytochemical localization of lysozyme and surfactant protein A in rat type II cells and extracellular surfactant forms.

Using immunogold labeling of fixed, cryosubstituted tissue sections, we compared the distribution of lysozyme, an oxidant-sensitive lamellar body protein, with that of surfactant protein A (SP-A) in rat Type II cells, extracellular surfactant forms, and alveolar macrophages. Morphometric analysis of gold particle distribution revealed that lysozyme and SP-A were present throughout the secretory and endosomal pathways of Type II cells, with prominent localization of lysozyme in the peripheral compartment of lamellar bodies. All extracellular surfactant forms were labeled for both proteins with preferential labeling of tubular myelin and unilamellar vesicles. Labeling of tubular myelin for SP-A was striking when compared with that of lamellar bodies and other extracellular surfactant forms. Lamellar body-like forms and multilamellar structures were uniformly labeled for lysozyme, suggesting that this protein is rapidly redistributed within these forms after secretion of lysozyme-laden lamellar bodies. By contrast, increased labeling for SP-A was observed over peripheral membranes of lamellar body-like forms and multilamellar structures, apparently reflecting progressive SP-A enrichment of these membranes during tubular myelin formation. The results indicate that lysozyme is an integral component of the lamellar body peripheral compartment and secreted surfactant membranes, and support the concept that lysozyme may participate in the structural organization of lung surfactant.     

A peculiar mode of formation of the surface lining layer in the lungs of Salamandra salamandra

The pneumocytes of the larva of Salamandra salamandra contain numerous lamellar bodies and their precursors: electron-dense bodies at various stages of development. Both lamellar bodies and electron-dense bodies occur inside the fluid-filled lung. The former are spherical or bell-shaped and possess concentrically arranged smooth membranes, 8 nm thick; the latter have paracrystalline cores composed of alternately oriented clear and dark striations (3.6–3.9 nm and 2.6–3.6 nm, respectively). On all sides such cores separate membranes, which assume a concentric orientation. No tubular myelin was observed in any phase of the transformation of lamellar bodies and electron-dense bodies into the surface lining layer. Fixation of the lungs of adult individuals with tannic acid-containing fixative visualized the surface lining layer, but not tubular myelin.     

Crystalloid structures within giant mitochondria of the prothoracic glands of Spodoptera littoralis (Bois) (Lepidoptera)

The fine structure of the prothoracic glands of Spodoptera littoralis was investigated during the first half of the last larval instar. The secretory cells have two types of mitochondria, micromitochondria and macromitochondria. The micromitochondria have rounded to elongated profiles and sometimes branch. They contain lamellar, tubular and also tubulo-vesicular cristae. The macromitochondria appear generally rounded or oval and possess tubular cristae. Many regular parallel membranes appear within macromitochondria. Favorable sections show tubular structures packed in honeycomb fashion. The mitochondrial cristae are in connection with the tubular structures. Honeycomb and parallel membranes increase in number as the size of the macromitochondria increases.     

Calcium-induced fusion of didodecylphosphate vesicles: The lamellar to hexagonal II (HII) phase transition

Summary Electron microscopic techniques have been employed to investigate the ability of didodecylphosphate vesicles (diameter approx. 900 Å) to fuse in the presence of Ca²⁺. As revealed by negative staining, Ca²⁺ induces extensive fusion and large vesicles with diameters up to 7000 Å are formed. In a processsecondary to fusion, the fused vesicles display a tendency to flatten and are subsequently transformed into extended tubular structures. Freeze-fracture electron microscopy, in conjunction with³¹P NMR and selected area electron diffraction measurements indicate that the tubes are packed in a hexagonal (H_II) array and that the amphiphiles are converted from the lamellar to the hexagonal H_II phase.The relationship between membrane fusion and the lamellar-to-hexagonal phase transition is discussed in terms of formation and abundance of transiently stable inverted micellar intermediates at contact regions between two interacting membranes. A model for the conversion of the (vesicular) lamellar into the (tubular) hexagonal H_II phase is presented, taking into account the molecular shape of the amphiphile. The relevance of using simple synthetic amphiphiles as models for phospholipid bilayers and complex biomembrane behavior is briefly discussed.     

Surfactant protein A is localized at the corners of the pulmonary tubular myelin lattice

Immunogold labeling on sections of a freeze-substituted tubular myelin-enriched fraction isolated from a bronchoalveolar lavage of rat lung showed that surfactant protein A (SP-A) occurs predominantly at the corners of the tubular myelin lattice. Seventy-nine percent of the gold particles were located within 20 nm from a corner. Extracellular SP-A was detected only in the tubular myelin lattice and not in vesicles or secreted lamellar bodies. Ultra-thin cryosections of rat lung fixed in vivo showed that intracellular SP-A was distributed homogeneously over the stacked membranes of lamellar bodies in alveolar Type II cells. The presence of SP-A at the corners of the tubular myelin lattice suggests an important role of this protein in the formation and/or maintenance of this highly ordered lattice.     

Electron Tomography of Neuronal Mitochondria: Three-Dimensional Structure and Organization of Cristae and Membrane Contacts

The structure of neuronal mitochondria from chick and rat was examined using electron microscope tomography of chemically fixed tissue embedded in plastic and sliced in ≈500-nm-thick sections. Three-dimensional reconstructions of representative mitochondria were made from single-axis tilt series acquired with an intermediate voltage electron microscope (400 kV). The tilt increment was either 1° or 2° ranging from −60° to +60°. The mitochondrial ultrastructure was similar across species and neuronal regions. The outer and inner membranes were each ≈7 nm thick. The inner boundary membrane was found to lie close to the outer membrane, with a total thickness across both membranes of ≈22 nm. We discovered that the inner membrane invaginates to form cristae only through narrow, tubular openings, which we call crista junctions. Sometimes the cristae remain tubular throughout their length, but often multiple tubular cristae merge to form lamellar compartments. Punctate regions, ≈14 nm in diameter, were observed in which the inner and outer membranes appeared in contact (total thickness of both membranes ≈14 nm). These contact sites are known to a play a key role in the transport of proteins into the mitochondrion. It has been hypothesized that contact sites may be proximal to crista junctions to facilitate transport of proteins destined for the cristae. However, our statistical analyses indicated that contact sites are randomly located with respect to these junctions. In addition, a close association was observed between endoplasmic reticulum membranes and the outer mitochondrial membrane, consistent with the reported mechanism of transport of certain lipids into the mitochondrion.     

The fine structure of the mesonephros of the lamprey,Entosphenus japonicus Martens

Summary The fine structure of the mesonephric kidney of the lamprey, Entosphenus japonicus Martens, has been investigated with the electron microscope and discussed from the viewpoint of comparative morphology of the mesonephros.The structure of the capillary wall of the glomerulus essentially coincides with that of higher vertebrates, though its basement membrane is remarkably thick (300–400 m) because of a dense accumulation of fibrillar material between the endothelium and the basal lamina of epithelial cell. No obvious fenestration of the endothelial cell has been observed in the glomerulus or capillaries in any part of this organ.The kidney tubule is divided into three segments: 1. neck segment composed of ciliated cells with numerous mitochondria and glycogen particles, 2. proximal tubule composed of brush bordered cells provided with extensive pinocytotic vesicles and lysosomal granules in the apical cytoplasm and with lamellar membranes in the basal, and 3. distal tubule characterized by cells which, with their abundant mitochondria and branched tubular endoplasmic reticulum (about 500 Å diameter) with a central core, closely resemble the chloride cells in the gill filament of some teleosts. The possibility that the lamellar membranes in the proximal tubule cells correspond to basal infoldings is discussed.The extensive development of the tubular reticulum and of the mitochondria in the distal tubule cells is believed to reflect the active absorption of urine chloride in the urinary tubule of lamprey mesonephric kidney evidenced by physiologists. The proximal tubule is suggested to take a part also in the urinary transport of water and ions, as the lamellar membranes found in the cells of this portion likely correspond to the basal infoldings in more advanced forms of the kidney.The epithelial cells of the ureteric duct are characterized by granules suggesting a mucous secretion. No fine structure implying an absorptive activity in this duct has been observed.     

Evidence from solid-state NMR for nonhelical conformations in the transmembrane domain of the amyloid precursor protein

The amyloid precursor protein (APP) is subject to proteolytic processing by γ-secretase within neuronal membranes, leading to Alzheimer's disease-associated β-amyloid peptide production by cleavage near the midpoint of the single transmembrane (TM) segment of APP. Conformational properties of the TM segment may affect its susceptibility to γ-secretase cleavage, but these properties have not been established definitively, especially in bilayer membranes with physiologically relevant lipid compositions. In this article, we report an investigation of the APP-TM conformation, using ¹³C chemical shifts obtained with two-dimensional solid-state NMR spectroscopy as site-specific conformational probes. We find that the APP-TM conformation is not a simple α-helix, particularly at 37°C in multilamellar vesicles with compositions that mimic the composition of neuronal cell membranes. Instead, we observe a mixture of helical and nonhelical conformations at the N- and C-termini and in the vicinity of the γ-cleavage site. Conformational plasticity of the TM segment of APP may be an important factor in the γ-secretase cleavage mechanism.     

Selection of tunicamycin-resistant Chinese hamster ovary cells with increased N-acetylglucosaminyltransferase activity

Chinese hamster ovary (CHO) cells resistant to the antibiotic tunicamycin (TM) have been isolated by a stepwise selection procedure with progressive increments of TM added to the medium. TM inhibits asparagine-linked glycoprotein biosynthesis by blocking the transfer of N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to the lipid carrier. The TM-resistant cells exhibited a 200-fold increase in their LD50 for TM and were morphologically distinct from the parental cells. The rate of asparagine-linked glycoprotein biosynthesis was the same for wild-type and TM-resistant cells. Membrane preparations from TM-resistant cells cultured for 16 d in the absence of TM had a 15-fold increase in the specific activity of the UDP-N- acetylglucosamine:dolichol phosphate N-acetylglucosamine-1-phosphate transferase as compared to membranes of wild-type cells. The products of the in vitro assay were N-acetylglucosaminylpyrophosphoryl-lipid and N,N'-diacetylchitobiosylpyrophosphoryl-lipid for membranes from both TM- resistant and wild-type cells. The transferase activity present in membrane preparations from wild-type of TM-resistant cells was inhibited by comparable levels of TM. The data presented are consistent with overproduction of enzyme as the mechanism of resistance in these variant CHO cells.     

Anomalous Diffusion Induced by Cristae Geometry in the Inner Mitochondrial Membrane

Diffusion of inner membrane proteins is a prerequisite for correct functionality of mitochondria. The complicated structure of tubular, vesicular or flat cristae and their small connections to the inner boundary membrane impose constraints on the mobility of proteins making their diffusion a very complicated process. Therefore we investigate the molecular transport along the main mitochondrial axis using highly accurate computational methods. Diffusion is modeled on a curvilinear surface reproducing the shape of mitochondrial inner membrane (IM). Monte Carlo simulations are carried out for topologies resembling both tubular and lamellar cristae, for a range of physiologically viable crista sizes and densities. Geometrical confinement induces up to several-fold reduction in apparent mobility. IM surface curvature per se generates transient anomalous diffusion (TAD), while finite and stable values of projected diffusion coefficients are recovered in a quasi-normal regime for short- and long-time limits. In both these cases, a simple area-scaling law is found sufficient to explain limiting diffusion coefficients for permeable cristae junctions, while asymmetric reduction of the junction permeability leads to strong but predictable variations in molecular motion rate. A geometry-based model is given as an illustration for the time-dependence of diffusivity when IM has tubular topology. Implications for experimental observations of diffusion along mitochondria using methods of optical microscopy are drawn out: a non-homogenous power law is proposed as a suitable approach to TAD. The data demonstrate that if not taken into account appropriately, geometrical effects lead to significant misinterpretation of molecular mobility measurements in cellular curvilinear membranes.     

Fine structure of the corpus Luteum of the raccoon during pregnancy

Summary Ultrastructure of the granulosa lutein cells of the raccoon from throughout pregnancy has been described. The lutein cells often from epithelial cords which are separated by the connective tissues, capillaries and lymphatics. Based on the arrangements and modifications of the cytoplasmic organelles and inclusions, three types of lutein cells have been recognized. The type I lutein cells predominantly contain tubular, agranular endoplasmic reticulum, juxtanuclear Golgi complexes, a few round to rod-shaped mitochondria, some free ribosomes, and occasional lipid droplets. Occasionally the tubular cristae of mitochondria and tubular smooth endoplasmic reticulum appear contiguous. The type II cells contain abundant lace-like and/or stacked fenestrated endoplasmic reticulum cisternae that frequently form membranous whorls, some tubular, agranular endoplasmic reticulum, mitochondria, and lipid droplets. Mitochondria are usually small, but unusual large ones also occur. The small, rod-to round-shaped mitochondria usually have tubular cristae; but the large, oval, elongate, and cup shaped mitochondria possess tubular, lamellar, plate like, and whorl-like cristae. The plasma membranes of the cells are complexly elaborated and folded, especially when apposing each other. In favorable sections, strands of fenestrated cisternae appose the folds of the plasma membranes. In general, the amount of cytoplasmic organelles and inclusions vary greatly in the cells. The type III cells predominantly contain lipid droplets and sparse cytoplasmic organelles. The type I and II cells are found throughout pregnancy, but the type III cells are observed from mid gestation to term. The cytological features of type I and II cells suggest that they probably secrete most of the steroids, whereas the type III cells primarily store lipids.This research was supported by UPSHS grant AM-11376 and NIH contract 69-2136.     

Thermodynamic and kinetic stabilities of transmembrane helix bundles as revealed by single-pair FRET analysis: Effects of the number of membrane-spanning segments and cholesterol

The tertiary structures and conformational dynamics of transmembrane (TM) helical proteins are maintained by the interhelical interaction network in membranes, although it is complicated to analyze the underlying driving forces because the amino acid sequences can involve multiple and various types of interactions. To obtain insights into basal and common effects of the number of membrane-spanning segments and membrane cholesterol, we measured stabilities of helix bundles composed of simple TM helices (AALALAA)₃ (1TM) and (AALALAA)₃-G₅-(AALALAA)₃ (2TM). Association–dissociation dynamics for 1TM–1TM, 1TM–2TM, and 2TM–2TM pairs were monitored to compare stabilities of 2-, 3-, and 4-helical bundles, respectively, with single-pair fluorescence resonance energy transfer (sp-FRET) in liposome membranes. Both thermodynamic and kinetic stabilities of the helix bundles increased with a greater number of membrane-spanning segments in POPC. The presence of 30 mol% cholesterol strongly enhanced the formation of 1TM–1TM and 1TM–2TM bundles (~ ? 9 kJ mol^?1), whereas it only weakly stabilized the 2TM–2TM bundle (~ ? 3 kJ mol^?1). Fourier transform infrared-polarized attenuated total reflection (ATR-FTIR) spectroscopy revealed an ~30° tilt of the helix axis relative to bilayer normal for the 1TM–2TM pair in the presence of cholesterol, suggesting the formation of a tilted helix bundle to release high lateral pressure at the center of cholesterol-containing membranes. These results demonstrate that the number of membrane-spanning segments affects the stability and structure of the helix bundle, and their cholesterol-dependences. Such information is useful to understand the basics of folding and assembly of multispanning TM proteins.     

Biogenesis of lamellar bodies,lysosome-related organelles involved in storage and secretion of pulmonary surfactant

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.     

Electron microscopic study of subcapsular cell hyperplasia in the adrenal glands of IQI/Jic mice.

Hyperplastic cells in subcapsular cell hyperplasia (SCH) lesion in adrenal glands of female IQI/Jic mice were examined by electron microscopy. These cells were small and polygonal, and had irregular nuclei, elongated mitochondria with lamellar cristae and dense lipid droplets. While these cells showed different features, some of them had desmosomes and basement membranes, and a few round mitochondria with tubular cristae as endocrine cells. These findings suggest that hyperplastic cells in SCH lesions might originate from endocrine blastemic cells.     

The case for a polyphyletic origin of mitochondria: Morphological and molecular comparisons

The comparative morphology and pigmentation of protists suggest that those with tubular mitochondrial cristae belong to a different lineage than those with lamellar cristae and that the evolutionary divergence might have been very early. We propose that the difference in cristal morphology is the result of separate origins of the mitochondria from endosymbionts related to the Rhodospirillaceae (purple nonsulfur bacteria) but differing in the morphology of their internal membranes. Comparisons of the cytochromes c of protists and the Rhodospirillaceae and of 16s rRNA T1 oligonucleotide catalogs in the Rhodospirillaceae do not contradict, and in fact provide support for, the idea. More extensive evidence may be lacking simply because cytochromes c have been studied in very few protists with tubular mitochondrial cristae.     

The internal structure of mitochondria

Electron microscopic (EM) tomography is providing important new insights into the internal organization of mitochondria. The standard baffle model for cristae structure, called into question years ago, has now clearly been shown to be inaccurate. Depending on source and conformational state, cristae can vary from simple tubular structures to more complex lamellar structures merging with the inner boundary membrane through tubular structures 28 nm in diameter. The structural information provided by EM tomography has important implications for mitochondrial bioenergetics, biogenesis and the role of mitochondria in apoptosis. The structural paradigm defined by EM tomography is helping in the design of new experimental approaches to mitochondrial function.