Biophysicochemical Interaction of a Clinical Pulmonary Surfactant with Nanoalumina.

We report on the interaction of pulmonary surfactant composed of phospholipids and proteins with nanometric alumina (Al2O3) in the context of lung exposure and nanotoxicity. We study the bulk properties of phospholipid/nanoparticle dispersions and determine the nature of their interactions. The clinical surfactant Curosurf, both native and extruded, and a protein-free surfactant are investigated. The phase behavior of mixed surfactant/particle dispersions was determined by optical and electron microscopy, light scattering, and zeta potential measurements. It exhibits broad similarities with that of strongly interacting nanosystems such as polymers, proteins or particles, and supports the hypothesis of electrostatic complexation. At a critical stoichiometry, micron-sized aggregates arising from the association between oppositely charged vesicles and nanoparticles are formed. Contrary to the models of lipoprotein corona or of particle wrapping, our work shows that vesicles maintain their structural integrity and trap the particles at their surfaces. The agglomeration of particles in surfactant phase is a phenomenon of importance that could change the interactions of the particles with lung cells.

Mechanisms of protein sorting and coat assembly: insights from the clathrin-coated vesicle pathway.

Clathrin-coated vesicles have provided the best example illustrating how both soluble and membrane proteins are selectively clustered into a transport intermediate for subsequent delivery to another intracellular compartment. Like cytosolic clathrin adaptors, the adaptor-like complex AP-3 binds to specific membranes and selects membrane proteins by interacting with their sorting signals.

Roles for mannose-6-phosphate receptors in lysosomal enzyme sorting, IGF-II binding and clathrin-coat assembly.

Two related transmembrane proteins in mammalian cells bind the mannose 6-phosphate recognition marker present on all soluble lysosomal hydrolases. Recent studies of cells that express only one or neither of these proteins have shed light not only on their function in directing lysosomal enzymes into the endocytic pathway but also on their critical role in transport vesicle formation in the trans Golgi network. One of these proteins also binds insulin-like growth factor II (IGF-II) and may be an important part of the IGF-dependent system that regulates development.

Mannose 6-phosphate receptors regulate the formation of clathrin-coated vesicles in the TGN.

The transport of the two mannose 6-phosphate receptors (MPRs) from the secretory pathway to the endocytic pathway is mediated by carrier vesicles coated with the AP-1 Golgi-specific assembly protein and clathrin. Using an in vitro assay that reconstitutes the ARF-1-dependent translocation of cytosolic AP-1 onto membranes of the TGN, we have previously reported that the MPRs are key components for the efficient recruitment of AP-1 (Le Borgne, R., G. Griffiths, and B. Hoflack. 1996. J. Biol. Chem. 271:2162-2170). Using a polyclonal antibody against the mouse gamma-adaptin, we have now examined the steady state distribution of AP-1 after subcellular fractionation of mouse fibroblasts lacking both MPRs or reexpressing physiological levels of either MPR. We report that the amount of AP-1 bound to membranes and associated with clathrin-coated vesicles depends on the expression level of the MPRs and on the integrity of their cytoplasmic domains. Thus, these results indicate that the concentration of the MPRs, i.e., the major transmembrane proteins sorted toward the endosomes, determines the number of clathrin-coated vesicles formed in the TGN.

Diet of pre-settlement larvae of coral-reef fishes: selection of prey types and sizes.

This study examines the diet of nine taxa from seven families of pre-settlement coral-reef fish larvae collected in the lagoon of New Caledonia, south-west Pacific. Chesson's index of electivity indicated that the major prey groups in the diets, i.e. small copepods, small unidentified crustaceans and eggs, were positively selected by all larvae, provided they were at least 150 microm wide. This result emphasizes the role of larvae's behaviour on their feeding.

Protein transport from the secretory to the endocytic pathway in mammalian cells.

The trans-Golgi network (TGN) is the last station of the secretory pathway where soluble and membrane proteins are sorted for subsequent transport to endocytic compartments. This pathway is primarily followed by two distinct but related mannose 6-phosphate receptors which exhibit complementary functions in soluble lysosomal enzyme targeting. These transmembrane proteins and their bound ligands are packaged in transport intermediates coated with clathrin and the AP-1 assembly complex. Their segregation is determined by the interaction of tyrosine- and di-leucine-based sorting determinants present in their cytoplasmic domains with AP-1. Other membrane proteins such as the lysosomal membrane glycoproteins or envelope glycoproteins of herpes viruses, which contain similar sorting signals, may also follow the same pathway. In this review, we will summarize our current understanding of the molecular mechanisms leading to membrane protein sorting in the TGN and the formation of AP-1-coated transport intermediates.

Chimeric proteins containing the cytoplasmic domains of the mannose 6-phosphate receptors codistribute with the endogenous receptors.

We have constructed and transiently expressed in HeLa cells a series of hybrid proteins in which the cytoplasmic domain or both the transmembrane and the cytoplasmic domains of the mannose 6-phosphate/insulin-like growth factor II receptor were fused to the ectodomain of the hemagglutinin of the influenza virus (HA), a typical plasma membrane protein. In addition, we have expressed a hybrid protein containing the luminal domain of HA fused to the transmembrane and cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor. These hybrids were transported through and sorted from the secretory pathway as shown by acquisition of endo-H resistant oligosaccharides and their ability to recruit the Golgi assembly proteins AP-1 on the Golgi membrane. Like the mannose 6-phosphate receptors (MPRs), these hybrid proteins are also present in small amounts at the cell surface where they are likely to undergo endocytosis as disruption of the endocytosis signals contained in the MPR cytoplasmic domains induces their accumulation at the cell surface. Double immunofluorescence studies indicate that these chimeras codistribute with the endogenous MPRs at steady state. The results suggest that the cytoplasmic domains of the MPRs are sufficient to determine the steady-state distribution of the full-length proteins.

Spatial and temporal distribution of zooplankton related to the environmental conditions in the coral reef lagoon of New Caledonia, Southwest Pacific.

The distribution of zooplanktonic prey of fish larvae was examined in three bays and two lagoonal stations in the Southwest lagoon of New Caledonia. Water column conditions were characterized by increasing chlorophyll a and particulate organic matter (POM) concentrations from the lagoon to the estuarine bay. The mean zooplankton settled volume and total density were significantly higher in the estuarine bay, reaching 35.1 mL m(-3) and 3.5 x 10(5) individuals m(-3), respectively. The total zooplankton density also progressively increased along the sampling period. The composition of assemblages differed between the lagoon and the bays, and was similar in the three bays. Wind speed, surface temperature, chlorophyll a and POM explained these variations, as revealed by a co-inertia analysis (COIA). The prey preferred by fish larvae, i.e. small crustaceans and small copepods, were more abundant in bays. Sheltered bays, most influenced by terrigenous inputs, are likely to provide the best feeding conditions.

Mannose 6-phosphate receptors and ADP-ribosylation factors cooperate for high affinity interaction of the AP-1 Golgi assembly proteins with membranes.

Clathrin coat assembly in the trans-Golgi network, leading to the sequestration of the mannose 6-phosphate receptors (MPRs) into nascent vesicles, requires the ARF-1-dependent translocation of the cytosolic AP-1 Golgi assembly proteins onto the membranes of this organelle. The mechanistic role of the MPRs, i.e. the cargo molecules, in coat assembly is at present unclear. Using a GTP-dependent, brefeldin A-sensitive in vitro AP-1 binding assay, we have determined here the parameters of the AP-1 binding reaction. We demonstrate that, in addition of ARF-1, the MPRs contribute to create high affinity AP-1 binding sites (Kd approximately 25 mM), since their number correlates the number of MPR molecules expressed in MPR-negative cells. The quantitative electron microscopy shows that these high affinity binding sites are present on trans-Golgi network membranes, as expected, and to some extent on early endosomes. The high affinity binding sites are lost when the MPRs or ARF-1 become rate-limiting components. Conversely, GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)), which increases the amount of membrane-bound ARF-1, most uncovers low affinity AP-1 binding sites (Kd approximately 150 nM) on trans-Golgi network membranes, normally not detected in its absence. Collectively, these results argue that MPR sorting is highly coupled to the first step of coat assembly and that the MPRs, ARF-1, and possibly other proteins cooperate for high affinity interactions of AP-1.

The mammalian AP-3 adaptor-like complex mediates the intracellular transport of lysosomal membrane glycoproteins.

In mammalian cells, the mannose 6-phosphate receptors (MPRs) and the lysosomal glycoproteins, lysosomal-associated membrane protein (LAMP) I, lysosomal integral membrane protein (LIMP) II, are directly transported from the trans-Golgi network to endosomes and lysosomes. While MPR traffic relies on the AP-1 adaptor complex, we report that proper targeting of LAMP I and LIMP II to lysosomes requires the AP-3 adaptor-like complex. Overexpression of these proteins, which contain either a tyrosine- or a di-leucine-based-sorting motif, promotes AP-3 recruitment on membranes. Inhibition of AP-3 function using antisense oligonucleotides leads to a selective misrouting of both LAMP I and LIMP II to the cell surface without affecting MPR trafficking. These results provide evidence that AP-3 functions in the intracellular targeting of transmembrane glycoproteins to lysosomes.

Basolateral sorting of the cation-dependent mannose 6-phosphate receptor in Madin-Darby canine kidney cells. Identification of a basolateral determinant unrelated to clathrin-coated pit localization signals.

In polarized Madin-Darby canine kidney (MDCK) cells, sorting of membrane proteins in the trans-Golgi network for basolateral delivery depends on the presence of cytoplasmic determinants that are related or unrelated to clathrin-coated pit localization signals. Whether these signals mediate basolateral protein sorting through common or distinct pathways is unknown. The cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor (CD-MPR) contains clathrin-coated pit localization signals that are necessary for endocytosis and lysosomal enzyme targeting. In this study, we have addressed the function of these signals in polarized sorting of the CD-MPR. A chimeric protein, made of the luminal domain of the influenza virus hemagglutinin fused to the transmembrane and cytoplasmic domains of the CD-MPR was stably expressed in MDCK cells. This chimera (HCD) is able to interact with the AP-1 Golgi-specific assembly proteins and is detected on the basolateral plasma membrane of MDCK cells where it is endocytosed. Deletion analysis and site-directed mutagenesis of the cytoplasmic domain of the CD-MPR indicate that HCD chimeras devoid of clathrin-coated pit localization signals are still transported to the basolateral membrane where they accumulate. A HCD chimera containing only the transmembrane domain and the 12 membrane-proximal amino acids of the CD-MPR cytoplasmic tail is also found on the basolateral membrane but is unable to interact with the AP-1 assembly proteins. However, the overexpression of this mutant results in partial apical delivery. It is concluded, therefore, that the basolateral transport of this chimera requires a saturable sorting machinery distinct from AP-1.

Ezrin has properties to self-associate at the plasma membrane.

Ezrin, a member of a family of proteins involved in the interaction of the microfilament cytoskeleton with the plasma membrane, plays a role in membrane translocation in gastric parietal cells (Hanzel, D., Reggio, H., Bretscher, A., Forte, J. G. and Mangeat, P. (1991). EMBO J. 10, 2363-2373). Human ezrin was expressed in and purified from Escherichia coli. It possesses all the major biophysical, immunological and physiological properties of natural ezrin. Upon microinjection in live gastric HGT-1 cells, ezrin was incorporated into the dorsal microvilli, a site where the endogeneous protein is localized. By coimmunoprecipitation and ezrin-affinity assays, two HGT-1 cell proteins of 77 and 72 kDa behaved as ezrin-binding proteins. In enriched gastric apical membranes, 125I-ezrin labelled proteins of 80, 77 and 72 kDa by overlay assay. The 80 kDa protein was identified as ezrin and the 77 and 72 kDa proteins as gastric forms of proteins structurally related to ezrin, such as radixin and moesin. In insect cells infected with a recombinant baculovirus, one-third of over-expressed ezrin accumulated at the plasma membrane. Ezrin bound a 77 kDa endogenous peripheral membrane protein, behaving as an insect counterpart of the mammalian ezrin family. In addition to the respective role of the amino- and carboxyl-terminal domains of ezrin in linking the membrane and the cytoskeleton (Algrain, M., Turunen, O., Vaheri, A., Louvard, D. and Arpin, M. (1993). J. Cell Biol. 120, 129-139), both domains interacted synergistically in a salt-dependent manner to trigger self-association of ezrin. Ezrin's self-association properties could represent another way of regulating the number of ezrin molecules bound at specific membrane sites.

A casein kinase II phosphorylation site in the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor determines the high affinity interaction of the AP-1 Golgi assembly proteins with membranes.

The transport of proteins from the secretory to the endocytic pathway is mediated by carrier vesicles coated with the AP-1 Golgi assembly proteins and clathrin. The mannose 6-phosphate receptors (MPHs) are two major transmembrane proteins segregated into these transport vesicles. Together with the GTPase ARF-1, these cargo proteins are essential components for the efficient translocation of the cytosolic AP-1 onto membranes of the trans-Golgi network, the first step of clathrin coat assembly, MPR-negative fibroblasts have a low capacity of recruiting AP-1 which can be restored by re-expressing the MPRs in these cells. This property was used to identify the protein motif of the cation-dependent mannose 6-phosphate receptor (CD-MPR) cytoplasmic domain that is essential for these interactions. Thus, the affinity of AP-1 for membranes and in vivo transport of cathepsin D were measured for MPR-negative cells re-expressing various CD-MPR mutants. The results indicate that the targeting of lysosomal enzymes requires the CD-PDR cytoplasmic domain that are different from tyrosine-based endocytosis motifs. The first is a casein kinase II phosphorylation site (ESEER) that is essential for high affinity binding of AP-1 and therefore probably acts as a dominant determinant controlling CD-MPR sorting in the trans-Golgi network. The second is the adjacent di-leucine motif (HLLPM), which, by itself, is not critical for AP-1 binding, but is absolutely required for a downstream sorting event.

Binding of AP-1 Golgi adaptors to membranes requires phosphorylated cytoplasmic domains of the mannose 6-phosphate/insulin-like growth factor II receptor.

In mammalian cells, clathrin-coated vesicles mediate transport of the lysosomal enzyme receptors from the trans-Golgi network to the endocytic pathway. A critical step of this process is the recruitment of Golgi-specific adaptors onto Golgi membranes for efficient clathrin polymerization. An in vitro assay was used here to quantitate this event in streptolysin-O-permeabilized NRK cells. At 37 degrees C, these interactions are cytosol- and energy-dependent, sensitive to GTP gamma S (guanosine 5'-O-(thiotriphosphate)) and brefeldin A. We report that Golgi-specific adaptor binding is enhanced in mannose 6-phosphate/insulin-like growth factor II (IGF II) receptor-overexpressing cells and reduced in mannose 6-phosphate receptor-deficient cells. Furthermore, adaptor binding is partially inhibited after addition of soluble cytoplasmic domains of the mannose 6-phosphate/IGF II receptor. Almost complete inhibition is only observed when this domain is phosphorylated on serines 2421 and 2492, a major modification acquired during exit of the receptor from the Golgi. These results show that the mannose 6-phosphate/IGF II receptor is part of the components that recruit the Golgi-specific adaptors and that its phosphorylation is an important feature for high affinity interactions with sorting components.

Expression of major histocompatibility complex class II molecules in HeLa cells promotes the recruitment of AP-1 Golgi-specific assembly proteins on Golgi membranes.

The newly synthesized major histocompatibility complex (MHC) class II molecules, an alphabeta dimer associated with the Ii invariant chain, must be targeted to endosomal, lysosomal enzyme-rich compartments in order to bind and present immunogenic peptides. The precise route followed by this complex at the exit of the trans-Golgi network, the last sorting station of the biosynthetic pathway, is poorly understood. We show here that overexpression of alphabetaIi complexes in HeLa cells promotes the first step of clathrin-coat assembly in vitro, that is the ARF-dependent translocation of AP-1 Golgi-specific assembly proteins on membranes. In contrast, alphabeta dimers alone or associated with Ii lacking most of its cytoplasmic domain fail to recruit AP-1. This study strongly suggests that the invariant chain (Ii) is responsible for the AP-1-dependent sorting of the alphabeta dimers in the trans-Golgi network of HeLa cells and that the MHC class II molecules are, like the mannose 6-phosphate receptors, transported directly from this compartment to endosomes via clathrin-coated vesicles.

The AP-3-dependent targeting of the melanosomal glycoprotein QNR-71 requires a di-leucine-based sorting signal.

The Quail Neuroretina clone 71 gene (QNR-71) is expressed during the differentiation of retinal pigmented epithelia and the epidermis. It encodes a type I transmembrane glycoprotein that shares significant sequence homologies with several melanosomal proteins. We have studied its intracellular traffic in both pigmented and non-pigmented cells. We report that a di-leucine-based sorting signal (ExxPLL) present in the cytoplasmic domain of QNR-71 is necessary and sufficient for its proper targeting to the endosomal/premelanosomal compartments of both pigmented and non-pigmented cells. The intracellular transport of QNR-71 to these compartments is mediated by the AP-3 assembly proteins. As previously observed for the lysosomal glycoproteins Lampl and LimpII, overexpression of QNR-71 increases the amount of AP-3 associated with membranes, and inhibition of AP-3 synthesis increases the routing of QNR-71 towards the cell surface. In addition, expression of QNR-71 induces a misrouting of endogenous LampI to the cell surface. Thus, the targeting of QNR-71 might be similar to that of the lysosomal integral membrane glycoproteins LampI and LimpII. This suggests that sorting to melanosomes and lysosomes requires similar sorting signals and transport machineries.

Effects of discrete lesions in the ventral noradrenergic ascending bundle on the corticotropic stress response depend on the site of the lesion and on the plasma levels of adrenal steroids.

Stereotaxic deletion of selected areas of the ventral noradrenergic ascending bundle (VNAB-X) by discrete bilateral injections of 6-hydroxydopamine (6-OHDA; 4 micrograms in 0.2 microliter saline) was used to explore the role of brain catecholamines (CA) and their interaction with corticosteroid feedback in stress responses of the ACTH-corticosterone (CORT) axis. The stereotaxic coordinates used for 6-OHDA lesions and the optimization of postlesion delays were determined by (a) radioautographic labeling of the VNAB axons after tracer injections into the dorsal A2/C2, or the ventral A1/C1 medullary areas, (b) histofluorescence and immunocytochemical location of interrupted CA pathways versus the postlesional scar, and (c) postlesional noradrenaline and adrenaline concentrations in whole hypothalami and paraventricular nuclei (PVN) punch samples. Two sites of 6-OHDA lesions were selected; both led to striking falls in PVN concentrations of both CA. The more dorsal lesion (dVNAB-X) was the same as that of our earlier studies and interrupted pathways originating predominantly in the A2/C2 area; the second more ventral lesion (vVNAB-X) interrupted axons stemming preferentially from the A1/C1 area. Both VNAB lesions inhibited the ether stress-induced ACTH and CORT surges in rats with intact adrenals. But the blockade (overall poststress release, amplitude and swiftness of hormonal responses) by dVNAB-X was greater than by vVNAB-X. The basal ACTH level in adrenalectomized rats (ADX) was elevated 20-fold and ether stress induced a 4-fold ACTH surge. As in sham-ADX rats, vVNAB-X in ADX rats induced only moderate inhibition of the ACTH response versus ADX + sham-vVNAB-X controls. On the other hand ADX + dVNAB-X rats showed a greatly amplified ACTH stress response over the ADX-sham dVNAB-X controls. This amplification was reversed by oral CORT supplementation. The data suggest that the CA pathways of the VNAB participating, directly or indirectly, in the poststress corticotropic activation may include subsets of CA axons of different origins, whose functional roles in stress are modulated in opposite directions by the plasma corticosteroid level.