Dictyostelium discoideum cells shed vesicles with associated DNA and vital stain Hoechst 33342.

Dictyostelium discoideum cells are highly resistant to xenobiotics. We previously observed that these primitive eukaryotic cells contain a 170-kDa P-glycoprotein, mediating multidrug resistance in mammalian cells, but nonfunctional in Dictyostelium cells. We show here that D. discoideum cells vitally stained with the DNA-specific dye, Hoechst 33342, release fluorescent material in their culture medium. Electron microscopy and lipid analysis demonstrate the vesicular nature of this material. Moreover, nucleic acids associate with these extracellular vesicles independently of Hoechst vital staining. The main vesicular DNA component exhibits a size > 21 kb. Shedding of microvesicles during cell growth is not concomitant with programmed cell death. We propose that these extracellular vesicles are involved in a new cellular resistance mechanism against xenobiotics. Furthermore, since the association of DNA with vesicles occurs in physiological growth conditions and independently of vital staining, the new shedding process might be involved in a more general intercellular mechanism.

In adrenocortical tissue, annexins II and VI are attached to clathrin coated vesicles in a calcium-independent manner.

We have previously characterized three populations of clathrin coated vesicles (CCVs) isolated from bovine adrenocortical tissue and designated them as large, medium and small coated vesicles, i.e., LCV, MCV and SCV, respectively. Here, we show that annexins II and VI, two of the annexins involved in membrane traffic, are present in the three populations of CCVs but with different distributions between coat proteins (CP) and lipidic vesicle membrane. Annexin VI is only associated with the membrane, whatever the CCV population. In contrast, annexin II is differently distributed between coat and membrane, depending on the CCV population. Both annexins are bound to membranes in a calcium-independent manner and solubilization studies in Triton X114 (TX114) suggest that they interact poorly with lipids by hydrophobic interactions. Ligand blotting experiments show that both annexins bind to CCV proteins: annexin II to a 200-kDa component in all CCVs and annexin VI to a 100-kDa component in LCV and SCV identified as dynamin, a GTPase essential for endocytic CCV pinching off. Dynamin is tightly associated to annexin VI only in LCVs, the endocytic [transferrin (Tf) positive] vesicles. Our data suggest that annexins II and VI could define specific protein-lipid interaction microdomains that could play a role in the different functions of the CCVs.

Specific lipid protein interactions characterize 3 populations of clathrin coated vesicles involved in the LDL receptor traffic.

We have previously isolated 3 different populations of clathrin coated vesicles (CCV) involved in the LDL-receptor traffic in bovine adrenal cortex. We now show that each CCV type contains the transferrin-R and the CI-MPR, therefore, they provide a good model for studying the membrane organization that may govern their targeting in one of the biosynthetic, endocytic and/or recycling pathways. Transferrin--prototype of recylcing ligand--, and alpha adaptin, dynamin and the 110 kDa phosphatidylinositol-3-kinase subunit--of the trafficking machinery--were mainly detected in only 2 of the vesicle populations which could be involved in the endocytic/recycling pathway. The third population which contained larger amounts of gamma adaptin and do not carry transferrin could be involved in the biosynthetic pathway. The vesicle lipid pattern and the saturation of their fatty acyl chains were analyzed and confirmed these results. The nature of the interactions between vesicle components was then determined using several classes of detergents. Only non ionic ones could solubilize the LDL-R in a complex with either alpha or gamma adaptin. In contrast, they dissociated clathrin or beta-beta' adaptins. Taken together these results prompt us to suggest an integrated model for targeting in membrane traffic. Besides specific targeting signals carried by cargo proteins and recognized by proteins from the coat and the cytosolic trafficking machinery, lipids would play a key modulatory role. At each step in the membrane traffic, the proteins which carry multiple targeting signals would interact transiently with a specific set of lipids. This would result in the exposure of the appropriate targeting signals which could now become recognized by the proper targeting machinery.

Biochemical characterization of algal coated vesicles.

Thin sections of tissue preparations from a green alga, Ulva lactuca (Ulvophyceae), and brown alga, Laminaria digitata (Pheophyceae) showed the presence of coated pits and coated vesicles in these 2 species. A discontinuous sucrose gradient after subcellular fractionation of the tissue homogenate resulted in an enriched coated vesicle fraction. Electron microscopy of negatively stained samples revealed the presence of coated vesicles of diameter ranging from 40-125 nm, together with large sheets of polygonal nets of clathrin. Electrophoresis of the CV purified fraction revealed various polypeptide components. Two of them, a 175 kDa and a 70 kDa, exhibited a positive response to bovine brain anticlathrin antibodies raised in goat or in rabbit. A third component of 30-40 kDa also gave a faint positive response. These 3 components corresponded to the clathrin heavy and light chains already described in higher plants. Clathrin was released from the CV algal preparations by treatment with 2M urea in Tris buffer, pH 8.5. Interestingly, in Ulva lactuca, the proportion of clathrin relative to the other proteins from the CV decreased with plant growth. Biochemical analysis of the purified CV revealed the presence of all the major phospholipids characterized in mammalian CV. The ratio of protein over lipid was also in the same range as that calculated for mammalian CV. Carbohydrate analysis demonstrated a high proportion of N-acetylgalactosamine and N-acetylglucosamine in both algal CV whereas these sugars were not detectable in the crude homogenate. These results demonstrate the presence of clathrin and coated vesicles in 2 species of algae.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical and functional characterization of three types of coated vesicles in bovine adrenocortical cells: implication in the intracellular traffic.

Three populations of pure coated vesicles from adrenocortical cells, differing in their density, i.e., 1.125-1.155, 1.155-1.175, and 1.175-1.210 g/cm3, are obtained after separation on two successive sucrose-2H2O gradients. They are involved in LDL internalization and in the receptor cycle as confirmed by the presence, in each population, of the LDL receptor. Electron micrographs confirm the existence of three homogeneous populations exhibiting the typical polygonal structure of the clathrin coat. They differ in their size distribution (small, congruent to 70-nm diameter; medium, congruent to 90-nm diameter; large, congruent to 110-nm diameter) and in the organization of clathrin and of the coat proteins as evidenced on electrophoreses carried out under nondenaturing and denaturing conditions. Activity measurements of marker enzymes, phosphodiesterase and galactosyltransferase, suggest that medium coated vesicles might originate from plasma membranes and small ones from the Golgi complex. Large coated vesicles exhibit phosphokinase enzyme and substrate polypeptides different from those of the two other populations, tubulins being the preferred kinase substrates for the small and medium coated vesicles. These kinases are autophosphorylating enzymes and are revealed, by nondenaturing electrophoreses, as different high molecular mass complexes in the three populations. Clathrin and coat proteins are not part of these complexes.

Clustering in coated vesicles of polyunsaturated phospholipids segregated from plasma and Golgi membranes of adrenocortical cells.

In bovine adrenocortical cells, the fatty acyl chains of the phospholipids have been identified in the membranes of the different cell compartments: plasma membranes, Golgi complex and coated vesicle membranes. An increase in the total number of unsaturation in the fatty acid is demonstrated in the coated vesicle membranes as compared with the plasma and Golgi membranes. Furthermore, it appears that phosphatidylcholine and phosphatidylethanolamine are both enriched in polyunsaturated fatty acyl chains, namely arachidonic and adrenic acids in both types of coated vesicles. Only two of the fatty acids are characteristic of Golgi complex and small coated vesicles, 22:5 (n-6) in PC and 22:6 (n-3) in PE, suggesting that the SCV could originate from the Golgi stacks. A high value of the ratio 22:5 (n-3)/22:6 (n-3) is observed which is, as far as we know, characteristic of adrenal cells.

[Homology between antigenic determinants of bovine clathrin and clathrin from the brown alga Laminaria digitata]. / Homologie entre des déterminants antigéniques de clathrine de boeuf et de clathrine d'une algue brune Laminaria digitata.

Coated vesicles, essential organelles of intracellular membrane traffic, have been extensively studied in animal and higher plant cells. In the algae, cytological studies only have been performed which demonstrate the presence of such coated vesicles with their surrounding clathrin lattice. The present work has been carried out on coated vesicles isolated for the first time from the brown algae Laminaria digitata. For comparison of the antigenic characteristics of clathrin prepared from the Bovine brain or adrenocortical cells and the clathrin prepared from algae, polyclonal antibodies have been raised to a purified Bovine brain clathrin in Goat and to Bovine adrenocortical clathrin in Rabbit. The positive immunological responses of the coated vesicles and the clathrin from Algae to these antibodies, evidence an homology between antigenic determinants of clathrin from animal and vegetal cells.

Lipid bilayer dynamics in plasma and coated vesicle membranes from bovine adrenal cortex. Evidence of two types of coated vesicle involved in the LDL receptor traffic.

Pure coated vesicles have been prepared from the bovine adrenal cortex and two homogeneous populations have been separated, one of large diameter (100 nm) and one of small diameter (70 nm). The chemical composition in lipids and proteins of coated vesicles has been compared with that of partially purified plasma membranes and evidences a higher protein/lipid ratio and a higher concentration in phosphatidylethanolamine and unsaturated fatty acids. Evaluation of the lateral diffusion of pyrene in the lipid bilayer of coated vesicles as compared to uncoated vesicles evidences a slowing-down effect of clathrin. Measurements of lipids' rotational diffusion by time-resolved fluorescence indicate a decrease in the order parameter of the lipids in the coated vesicles due to clathrin. A hypothesis is proposed for a possible role of the clathrin coat in the concerted motion of lipids and proteins toward coated pits and in the mechanism of formation of coated vesicles. Separation of the large from the small coated vesicles made it possible to reveal different protein components in the two types of vesicle by electrophoresis and autoradiograms of the [gamma-32P]adenosine triphosphate- (ATP-) treated vesicles. Visualisation of the low-density lipoprotein receptor by ligand blotting and enzyme-linked immunosorbent assay (ELISA) techniques indicates an increased low-density lipoprotein receptor binding capacity in small coated vesicles as compared to large ones and plasma membranes.

Ligand-blotting visualization of the LDL receptor in plasma membranes and in two classes of coated vesicles from adrenocortical cells.

Two populations of coated vesicles, different in size, have been isolated from the bovine adrenal cortex. The enrichment of the LDL receptor from the plasma membrane to the large coated vesicles and then to the small ones was evidenced by ligand-blotting ELISA assays. The LDL receptor has been characterized as a 130-kDa proteic component which retains the binding specificity and structural features in plasma membranes as well as in the two classes of coated vesicles.

Effect of cholesterol and protein content on membrane fluidity and 3 beta-hydroxysteroid dehydrogenase activity in mitochondrial inner membranes of bovine adrenal cortex.

The steroid biosynthetic enzymes in the adrenal cortex are localised in endoplasmic reticulum and mitochondrial membranes. For some of the enzymes in endoplasmic reticulum the activity appears to be modulated by lipid fluidity, (21-hydroxysteroid hydroxylase and 3 beta-hydroxysteroid dehydrogenase). A mechanism for the regulation of corticosteroid biosynthesis mediated by the membrane fluidity has been suggested. Therefore a study of the mitochondrial inner membrane of the bovine adrenal cortex has been undertaken in comparison with a previous study of the endoplasmic reticulum. The kinetic parameters of the 3 beta-hydroxysteroid dehydrogenase were studied as a function of pH and temperature. No thermal transition can be observed in the Arrhenius plot for this enzyme in contrast with the results obtained for the microsomal enzyme. Membrane fluidity using, as fluorescent probes, diphenylhexatriene and a set of n-(9-anthroyloxy)fatty acids has been also studied as a function of temperature with or without addition of cholesterol. No thermal transition in the lipid phase can be observed. The addition of cholesterol to total mitochondrial membrane as to a lipid extract of the membrane decreases fluidity to the same extent as it does with microsomes. The presence of a large amount of protein in mitochondria has an effect which is additive to that of the cholesterol.

Organization and dynamics of lipids in bovine brain coated and uncoated vesicles.

Three characteristics have been demonstrated by the chemical analysis of bovine brain coated vesicles following removal of the coat proteins: a high protein content, a high cholesterol/lipid ratio and a high percentage of phosphatidylethanolamine amongst the phospholipids. The study of lipid bilayer organization and dynamics has been performed using the fluorescent probes pyrene and parinaric acid (cis and trans). This has allowed the study of both lateral mobility and rotational motion in the lipid bilayer of the coated and uncoated vesicles. Lateral mobility in the fluid phase of the lipid is slightly reduced by the presence of the clathrin coat, as indicated by the lower diffusion coefficient of pyrene in coated compared with uncoated vesicles. At all temperatures from 6 degrees to 30 degrees C, solid-phase domains, probed by trans parinaric acid, coexist with fluid-phase domains in the lipid bilayer. The temperature dependence of the parinaric acid lifetimes and of their amplitudes strongly suggests that the solid phase domains decrease in size with temperature, both in coated and uncoated vesicles. However, the difference in the value of the anisotropy at long times (r infinity), between coated and uncoated vesicles (a difference which is more pronounced for cis than for trans parinaric acid), indicates that the presence of the clathrin coat introduces disorder in the surrounding lipids, thus suggesting a possible role of the clathrin in the formation of the pits on the plasma membrane.

Solubilization and separation of delta5,3beta-hydroxysteroid dehydrogenase and 3-oxosteroid-delta4-delta5-isomerase from bovine adrenal cortex microsomes.

A physical separation of delta5,3beta-hydroxysteroid dehydrogenase and 3-oxosteroid delta4-delta5-isomerase solubilized from bovine adrenocortical microsomes is described for the first time. The solubilization as well as the separation was carried out with a mixture of a detergent: a substituted betaine (Empigen BB/P) and sodium cholate. This latter detergent protects isomerase from complete inactivation by Empigen and is necessary for the recovery of a significant amount of soluble isomerase. Separation of dehydrogenase and isomerase was successfully accomplished by the use of a DEAE-Biogel A anion-exchanger. Dehydrogenase activity was eluted, while the isomerase was retained. Measurements of dehydrogenase activity with androst-5-en-3beta-ol-17-one, pregnen-3beta-ol-20-one and pregn-5-en-(3beta,17alpha)-diol-20-one and of isomerase activity with androst-5-en-(3,17)-dione and pregn-5-en-(3,20)-dione suggested that more than one isomerase and more than one dehydrogenase form were present.

Lipid requirement of membrane-bound 3-oxosteroid delta4-delta5-isomerase. Studies on beef adrenocortical microsomes.

The role of phospholipid in the beef adrenal microsomal 3-oxosteroid delta4-delta5-isomerase (EC 5.3.1.1) has been investigated with the use of phospholipase A to alter the microsomal phospholipids. The byproducts of phospholipase A digestion have been removed with a wash solution containing bovine serum albumin. Removal of 80-85% of the phospholipid leads to loss of 80-90% of the 3-oxosteroid delta4-delta5-isomerase activity. Reconstitution experiments have been performed by introduction of lipid aqueous dispersions in the enzymatic assay. Asolectin, a commercially available preparation of soy phosphatides, is able to stimulate the enzymatic activity but does not restore the 3-oxosteroid delta4-delta5-isomerase activity in phospholipase-A-treated membranes. In contrast, the introduction of aqueous dispersions of microsomal total lipid mixtures in the enzymatic assay brings about a complete restoration of the 3-oxosteroid delta4-delta5-isomerase activity in the lipid-depleted membranes. It is concluded that the bovine adrenal microsomal 3-oxosteroid delta4-delta5-isomerase requires phospholipid(s) to exhibit its full catalytic activity.