Marlyse Zaepfel

The voltage-dependent anion channel, a major component of the tRNA import machinery in plant mitochondria

In plants, as in most eukaryotic cells, import of nuclear-encoded cytosolic tRNAs is an essential process for mitochondrial biogenesis. Despite its broad occurrence, the mechanisms governing RNA transport into mitochondria are far less understood than protein import. This article demonstrates by Northwestern and gel-shift experiments that the plant mitochondrial voltage-dependent anion channel (VDAC) protein interacts with tRNA in vitro. It shows also that this porin, known to play a key role in metabolite transport, is a major component of the channel involved in the tRNA translocation step through the plant mitochondrial outer membrane, as supported by inhibition of tRNA import into isolated mitochondria by VDAC antibodies and Ruthenium red. However VDAC is not a tRNA receptor on the outer membrane. Rather, two major components from the TOM (translocase of the outer mitochondrial membrane) complex, namely TOM20 and TOM40, are important for tRNA binding at the surface of mitochondria, suggesting that they are also involved in tRNA import. Finally, we show that proteins and tRNAs are translocated into plant mitochondria by different pathways. Together, these findings identify unexpected components of the tRNA import machinery and suggest that the plant tRNA import pathway has evolved by recruiting multifunctional proteins.

Receptor-mediated endocytosis of transthyretin by ependymoma cells

Transthyretin (TTR) is involved in the transport of thyroxine (T4) and retinol-binding protein (RBP) in cerebrospinal fluid (CSF) and serum. TTR is secreted in the CSF by the epithelial cells of choroid plexus. The binding of [(125)I]TTR to cultured ependymoma cells which form the brain cerebrospinal barrier, was studied to determine whether these cells carry receptor(s) for TTR. TTR was bound by ependymoma cells in a time-dependent manner reaching equilibrium within 2 h. Scatchard analysis was consistent with a single class of high-affinity binding sites with a K(d) of approximately 18 nM. Saturable high-affinity binding of human TTR has previously been described in rat primary hepatocytes and human renal adenocarcinoma, neuroblastoma, hepatoma and astrocytoma cells, and also transformed lung cells. Endocytosis of fluorescent or biotinylated TTR was observed in ependymoma cells in cytoplasmic vesicles but TTR did not colocalize with clathrin in endocytic coated vesicles. Endocytosis of TTR was inhibited by high sucrose concentration (0.45 M). Finally, ligand blotting and chemical-linking experiments revealed the presence of a approximately 100 kDa putative TTR receptor on the ependymoma cell membrane. Receptor binding of TTR provides a potential mechanism for the delivery of T4 within the central nervous system.

The presence of transthyretin in rat ependymal cells is due to endocytosis and not synthesis

The presence and synthesis of transthyretin, a major carrier protein of thyroxine in rat cerebrospinal fluid, was investigated in choroid plexus epithelial cells and ependymal cells by immunocytochemistry, in situ hybridization, and analysis by Northern and Western blot using a specific oligonucleotide probe and a specific polyclonal antibody to transthyretin. Choroid plexus epithelial cells expressed transthyretin at high levels in developing rat cerebral hemispheres and in cultured cells. These cells secreted transthyretin into the cerebrospinal fluid. In the developing rat brain transthyretin was present in the cytoplasm of ependymal cells, in vesicles in contact with the apical membrane and in cilia. In ependymal cell cultures this protein was particularly abundant in the cilia of these cells. In contrast, ependymal cells did not synthesize transthyretin. It is postulated that transthyretin is transported to ependymal cells from the cerebrospinal fluid by endocytosis.

Expression and localization in the developing cerebellum of the carbohydrate epitopes revealed by Elec-39, an IGM monoclonal antibody related to HNK-1

The immunochemical and immunocytochemical reactivity of an anti-carbohydrate monoclonal antibody (Elec-39), obtained against acetylcholinesterase from Electrophorus electricus electric organ, was followed during the postnatal development of the rat cerebellum. The specificity of this antibody resembles that of a family of anti-carbohydrate antibodies that includes HNK-1, L2, NC-1 and NSP-4, as well as IgMs that occur in some human neuropathies. As revealed by immunoblotting techniques, the reactivity of Elec-39 is maximum around postnatal days 10-12. At this age, the antibody reveals eight major proteins of mol. wt ranging between 14 and 150 kDa. Some of them (with mol. wts of 14, 18, 28 and 31 kDa) are transiently expressed. They correspond to previously identified glycoproteins binding to the plant lectin concanavalin A and binding also to the endogenous mannose-binding lectin CSL and endogenous membrane-bound mannose-binding lectin. In young animals, an important staining with the Elec-39 antibody can be observed on postmitotic precursors of granule cells, on astrocyte processes in the external granular layer, on newly formed parallel fibres and on unmyelinated axons of the white matter. In adult animals, the labelling is localized essentially in myelin and also in the cytoplasm of astrocytes. These results are discussed in relation to ontogenetic phenomena occurring during cerebellar development and the potential role of the carbohydrate epitope revealed with Elec-39 as a determinant in cell adhesion processes.

The Endogenous Lectin Cerebellar Soluble Lectin and Its Ligands in Central Nervous System Myelin of Myelin-Deficient (mld) Mutant Mice

Abstract: The myelin‐deficient (mld) mutation is an autosomal recessive mutation in the murine CNS exhibiting severe hypomyelination. The primary defect results in a drastic reduction of myelin basic protein synthesis caused by a duplication of the myelin basic protein gene with partial inversion of the upstream gene copy. The severe deficit of myelin basic protein is responsible for the absence of the major dense line but cannot explain the heterogeneity of myelin compaction found in mid. We have tested the hypothesis that the endogenous cerebellar soluble lectin (CSL) and/or its endogenous glycoprotein ligands could be involved in myelin abnormalities in the dysmyelinating mutant, mld. Immunocytochemical and immunoblotting techniques showed that the CSL level was not reduced significantly in the mld mutant. Furthermore, two ligands of CSL, the myelin‐associated glycoprotein and an axonal glycoprotein, with a relative molecular mass of 31 kDa, were not decreased in level in the purified myelin fraction isolated from mld mice. In contrast, three minor glycoprotein ligands of CSL of relative molecular mass of 23, 18, and 16 kDa were greatly reduced in content. The reduced concentration of these low‐molecular‐mass glycoproteins in mld myelin suggests that they are constituents of compact myelin. Furthermore, the observation that CSL is specifically localized in vivo in regions where mld myelin is more compact and absent from regions devoid of myelin compaction may suggest that the endogenous CSL lectin, as well as its minor glycoprotein ligands, plays a role in the stabilization of the myelin sheath.

Dexamethasone represses 3,5,3'-triiodothyronine-stimulated expression of intercellular adhesion molecule-1 in the human cell line ECV 304.

The effect of the thyroid hormone L-3,5,3′-triiodothyronine (T3) on the expression of ICAM-1, a cell surface glycoprotein playing a pivotal role in inflammatory responses, was investigated. In ECV 304 cells, T3 (30 nmol/L) markedly increased ICAM-1 protein expression, with a peak after 24 h of treatment. ECV 304 human cells express both α1 and β1 T3 receptors. In an attempt to understand the molecular mechanisms leading to the induction of the ICAM-1 gene by T3, we have studied the effects of a synthetic glucocorticoid, dexamethasone, and of a sesquiterpene lactone, parthenolide, on this T3-stimulated expression of ICAM-1. Our results demonstrate a repressive effect of dexamethasone and parthenolide on the expression of the protein ICAM-1 stimulated by T3. Both anti-inflammatory compounds interfere with this T3-mediated pathway in a dose-dependent manner.

Role of an Endogenous Mannosyl-Lectin in Myelination and Stabilization of Myelin Structure

The studies of the mechanisms involved in myelination and myelin compaction constitute a broad field of neurobiology since myelin plays an important role of in conductance of nerve impulse both in central and peripheral nervous system. One of the most dramatic demyelinating diseases is multiple sclerosis (MS). The study of demyelination in MS may help in understanding the molecular mechanisms involved in maintenance of myelin structure. Several hypothesis have been presented for explaining myelin compaction. From the studies of mid mutant (Doolittle and Schwiekart, 1977), it has been proposed (Jacque et al., 1983; Kimura et al., 1985; Lachapelle et al., 1980; Matthieu 1982; Matthieu et al., 1980a; 1980b; 1981; 1984; Mikoshiba et al., 1987; Quarles, 1984; Waehneldt and Linington, 1980) that myelin basic protein (MBP) is involved in the mechanism of myelin compaction at the cytoplasmic surface of the oligodendrocyte membrane. A similar role has been attributed to proteolipid protein (Duncan et al., 1987). Due to the specific defect of MBP in mld mutant there is an absence of the major dense line. It has also been proposed that this adhesion involved interaction of myelin basic protein with negatively charged glycolipids (i.e. sulfatides) present in high amount in these membranes (Zalc et al., 1981). Another possibility was the interaction of MBP with other glycolipids (Ikeda and Yamamoto, 1987). Similarly, the proteolipid protein (PLP) has been suggested to be involved in myelin compaction at the level of extracellular face of the oligodendrocyte (Dautigny et al., 1986).

Cerebellar soluble lectin and its glycoprotein ligands in the developing brain of control and dysmyelinating mutant mice

The levels of an endogenous lectin, the cerebellar soluble lectin (CSL) and of its endogenous glycoprotein ligands were studied using immunoblotting and affinoblotting techniques in the forebrain of quaking, shiverer and jimpy dysmyelinating mutant mice and their respective control littermates during the postnatal development. In the controls of the mutant mice, the level of CSL showed an important increase between days 5-18 then a stabilization, although at all ages the level of CSL was reduced (at least 15%) in the control littermate of the shiverer mutant. In the shiverer mutant the developmental pattern is similar to the control but was reduced by 50% as compared to the control. In the jimpy mutant an erratic development of CSL was observed which was with quasi absence of CSL at days 12 and 25. Variation of CSL levels in the quaking brain were also observed. CSL glycoprotein ligands also showed variable developmental profiles with a special persistence with ageing of CSL-binding glycoproteins in the quaking and jimpy mice. Developmental variations were also observed between the different control littermates. These results are discussed in view of developmental roles attributed to CSL and its glycoproteins ligands in cell adhesion mechanism during brain ontogenesis and especially myelination.

Endogenous mannose-binding lectins in brain development and function

The formation of brain tissue owes its specificity to multiple steps involving cell interactions during various stages of ontogenesis. Guided neuron migration, synaptogenesis and myelination constitute three essential steps in brain organization, where glycobiological recognition or adhesion systems play a fundamental role. The key molecules in these phenomena were postulated to belong to the family of cell adhesion molecules (CAMs; Crossin et al., 1986; Edelman, 1985 and 1986; Grumet et al., 1985; Hoffmann et al., 1986; Hoffmann and Edelman, 1987; Rieger et al., 1986; Salzer and Colman, 1989) acting by homophilic interactions. More recently two different mannose-binding endogenous lectins of similar specificity (Zanetta et al., 1985a and 1987a) seem to play important roles in relation with their endogenous glycoprotein ligands (Kuchler et al., 1988, 1989a,b and c; 1990a,b and c; Lehmann et al., 1990). It is worthy of mention that some of these glycoprotein ligands are members of the family of cell adhesion molecules (Kuchler et al., 1988, 1989a and b; 1990a and b) and share specific glycans binding to some onco-fetal antibodies. Thus, heterophilic interactions involving glycobiological system deserve attention. This paper summarizes the major observations concerned with the function in the brain of the two endogenous mannose-binding lectins, called CSL and R1 (Zanetta et al., 1985a and 1987a) and of their endogenous ligands.