Jean-Pierre Henry

Cloning of a functional vesicular GABA and glycine transporter by screening of genome databases

The unc‐47 locus of Caenorhabditis elegans has been suggested to encode a synaptic vesicle GABA transporter. Here we used hydropathy plot analysis to identify a candidate vesicular GABA transporter in genomic sequences derived from a region of the physical map comprising unc‐47. A mouse homologue was identified and cloned from EST database information. In situ hybridization in rat brain revealed codistribution with both GABAergic and glycinergic neuronal markers. Moreover, expression in COS‐7 and PC12 cells induced an intracellular, glycine‐sensitive GABA uptake activity. These observations, consistent with previous data on GABA and glycine uptake by synaptic vesicles, demonstrate that the mouse clone encodes a vesicular inhibitory amino acid transporter.

The GTPase Rab3a negatively controls calcium-dependent exocytosis in neuroendocrine cells.

There is accumulating evidence that small GTPases of the rab family regulate intracellular vesicle traffic along biosynthetic and endocytotic pathways in eukaryotic cells. It has been suggested that Rab3a, which is associated with synaptic vesicles in neurons and with secretory granules in adrenal chromaffin cells, might regulate exocytosis. We report here that overexpression in PC12 cells of Rab3a mutant proteins defective in either GTP hydrolysis or in guanine nucleotide binding inhibited exocytosis, as measured by a double indirect immunofluorescence assay. Moreover, injection of the purified mutant proteins into bovine adrenal chromaffin cells also inhibited exocytosis, as monitored by membrane capacitance measurements. Finally, the electrophysiological approach showed that bovine chromaffin cells which were intracellularly injected with antisense oligonucleotides targeted to the rab3a messenger exhibited an increasing potential to respond to repetitive stimulations. In contrast, control cells showed a phenomenon of desensitization. These results provide clear evidence that Rab3a is involved in regulated exocytosis and suggest that Rab3a is a regulatory factor that prevents exocytosis from occurring unless secretion is triggered. Furthermore, it is proposed that Rab3a is involved in adaptive processes such as response habituation.

Evolution of Reproductive Systems in the Genus Silene

The genus Silene contains both hermaphrodite, gynodioecious and dioecious species, dioecy being represented in three sections of the genus. To locate the events of change of reproductive systems, we compared ITS sequences of 22 species of Silene chosen throughout the whole genus, and four putative outgroup species. Gynodioecy, which is the most common reproductive system within the genus Silene and in closely related genera such as Saponaria and Dianthus, is proposed to be ancestral in the genus. Dioecy has evolved at least twice: once in the section containing S. latifolia, and once in the clade containing S. otites and S. acaulis ssp. bryoides. Evolution towards hermaphroditism, associated with evolution of selfing, has also occurred at least twice, in S. gallica and S. conica.

MyRIP, a novel Rab effector, enables myosin VIIa recruitment to retinal melanosomes

Defects of the myosin VIIa motor protein cause deafness and retinal anomalies in humans and mice. We report on the identification of a novel myosin‐VIIa‐interacting protein that we have named MyRIP (myosin‐VIIa‐ and Rab‐interacting protein), since it also binds to Rab27A in a GTP‐dependent manner. In the retinal pigment epithelium cells, MyRIP, myosin VIIa and Rab27A are associated with melanosomes. In transfected PC12 cells, overexpression of MyRIP was shown to interfere with the myosin VIIa tail localization. We propose that a molecular complex composed of Rab27A, MyRIP and myosin VIIa bridges retinal melanosomes to the actin cytoskeleton and thereby mediates the local trafficking of these organelles. The defect of this molecular complex is likely to account for the perinuclear mislocalization of the melanosomes observed in the retinal pigment epithelium cells of myosinVIIa‐defective mice.

Radioligands of the vesicular monoamine transporter and their use as markers of monoamine storage vesicles

A review on monoamine transporter of chromaffin granules, the catecholamine storage organelles of adrenal medulla chromaffin cells, using radioligands as a tool for the study of the monoamine uptake system of all monoaminergic vesicles

The vesiicular monoamine transporter: from chromaffin granule to brain

All characterized monoaminergic cells utilize the same transport system for the vesicular accumulation of monoamines prior to their release. This system operates in neuronal (catecholaminergic, serotoninergic or histaminergic) as well as in endocrine or neuroendocrine cells. For several decades, chromaffin granules from bovine adrenal medulla have been used as a model system, allowing progress in the understanding of the biophysics, the biochemistry and the pharmacology of the monoamine vesicular transporter. The transporters from rat, bovine and man have been cloned. Surprisingly, two genes encode different isoforms of the protein which are differentially expressed in monoaminergic systems. The conjunction of recombinant DNA techniques and expression in secretory or non-secretory cells with the large body of data obtained on the chromaffin granule transporter has allowed rapid progress in the study of the protein. But interestingly enough, this progress has open new possibilities in the study of biological problems, especially in the brain. The transporter is useful for the determination of the relationship between small and large dense core vesicles, for the understanding of the mechanism of the drugs such as 1-methyl-4-phenylpyridinium (MPP+), tetrabenazine or amphetamines, and as a marker in brain development. The possibility of regulations at the vesicular transporter level and of their effect on the quantum size has to be investigated. The vesicular monoamine transporter is also an important target for brain imaging.

Myosin Va Mediates Docking of Secretory Granules at the Plasma Membrane

Myosin Va (MyoVa) is a prime candidate for controlling actin-based organelle motion in neurons and neuroendocrine cells. Its function in secretory granule (SG) trafficking was investigated in enterochromaffin cells by wide-field and total internal reflection fluorescence microscopy. The distribution of endogenous MyoVa partially overlapped with SGs and microtubules. Impairing MyoVa function by means of a truncated construct (MyoVa tail) or RNA interference prevented the formation of SG-rich regions at the cell periphery and reduced SG density in the subplasmalemmal region. Individual SG trajectories were tracked to analyze SG mobility. A wide distribution of their diffusion coefficient, Dxy, was observed. Almost immobile SGs (Dxy < 5 × 10−4 μm2 · s−1) were considered as docked at the plasma membrane based on two properties: (1) SGs that undergo exocytosis have a Dxy below this threshold value for at least 2 s before fusion; (2) a negative autocorrelation of the vertical motion was found in subtrajectories with a Dxy below the threshold. Using this criterion of docking, we found that the main effect of MyoVa inhibition was to reduce the number of docked granules, leading to reduced secretory responses. Surprisingly, this reduction was not attributable to a decreased transport of SGs toward release sites. In contrast, MyoVa silencing reduced the occurrence of long-lasting, but not short-lasting, docking periods. We thus propose that, despite its known motor activity, MyoVa directly mediates stable attachment of SGs at the plasma membrane.

Expression and regulation of the bovine vesicular monoamine transporter gene

In monoaminergic cells, the neurotransmitter is accumulated into secretory or synaptic vesicles by a tetrabenazine‐ and reserpine‐sensitive transporter, catalyzing an H+/monoamine antiport. The major vesicular monoamine transporter from bovine chromaffin cells was cloned, using sequences common to adrenal medulla and brain rat vesicular monoamine transporters. Its identity was confirmed by peptide sequences, determined from the purified protein. Surprisingly, the bovine adrenal medulla sequence, bVMAT2, is more related to the transporter from human and rat brain than to that from rat adrenal medulla. PCR amplification showed that bVMAT2, is expressed in both adrenal medulla and brain, in contrast with the situation reported in rats, where distinct genes appear to be expressed in brain (SVAT or MAT, now renamed rVMAT2) and in the adrenal medulla (CGAT, now renamed rVMAT1). In bovine chromaffin cells, long‐term depolarization by KC1 resulted in an increase in the level of bVMAT2 mRNA, in agreement with the previously observed increase in the transporter binding sites, suggesting that a coupling between stimulation, secretion and synthesis changes the composition of the secretory granule membrane.

Bovine Adrenal Tyrosine Hydroxylase: Comparative Study of Native and Proteolyzed Enzyme, and Their Interaction with Anions

Abstract: The pH optimum of native adrenal medulla tyrosine hydroxylase activity is shifted from 5.8 to 6.4 by polyanions (heparin, dextran sulphate), salts (NaCl, Na2SO4) and the anionic buffer 2‐(N‐morpholino)ethanesulphonic acid (MES). Simultaneously, the activity at the optimal pH is increased. Kinetic studies have shown that this activation is associated with a decrease of the apparent Km of the enzyme for the cofactor 6,7‐dimethyltetrahydropterin (DMPH4) and an increase in the Vmax for tyrosine and DMPH4. The Km for the tyrosine remained unchanged. These data have been interpreted in terms of the polyelectrolyte theory. The adsorption of tyrosine hydroxylase on various affinity gels containing heparin, dextran sulphate or unsulphated polymer dextran as ligands indicate that the activation of the enzyme is mediated by electrostatic interactions with the anionic species. The site of electrostatic interaction possesses some specificity since the binding constants are higher for heparin or dextran sulphate than for NaCl or MES buffer. Moreover, 3‐(N‐morpholino)propanesulphonic acid (MOPS) a slightly structurally different buffer inhibits the enzyme activity whereas N‐(2‐acetamido)‐2‐amino‐ethanesulphonic acid (ACES) has no effect. A limited proteolytic digestion which preserves the enzymatic activity, destroys the effects of the anions. The isoelectric point and the molecular parameters of tyrosine hydroxylase are markedly altered after limited digestion. It is therefore suggested that the interaction between the hydroxylase and anionic compounds occurs on a part of the protein which is different from the active site and which is lost by proteolysis. This portion of the protein might be involved in regulation of native tyrosine hydroxylase.

Cholesterol regulates membrane binding and aggregation by annexin 2 at submicromolar Ca 2+ concentration

Annexin 2 is a member of the annexin family which has been implicated in calcium-regulated exocytosis. This contention is largely based on Ca(2+)-dependent binding of the protein to anionic phospholipids. However, annexin 2 was shown to be associated with chromaffin granules in the presence of EGTA. A fraction of this bound annexin 2 was released by methyl-beta-cyclodextrin, a reagent which depletes cholesterol from membranes. Restoration of the cholesterol content of chromaffin granule membranes with cholesterol/methyl-beta-cyclodextrin complexes restored the Ca(2+)-independent binding of annexin 2. The binding of both, monomeric and tetrameric forms of annexin 2 was also tested on liposomes of different composition. In the absence of Ca(2+), annexin 2, especially in its tetrameric form, bound to liposomes containing phosphatidylserine, and the addition of cholesterol to these liposomes increased the binding. Consistent with this observation, liposomes containing phosphatidylserine and cholesterol were aggregated by the tetrameric form of annexin 2 at submicromolar Ca(2+) concentrations. These results indicate that the lipid composition of membranes, and especially their cholesterol content, is important in the control of the subcellular localization of annexin 2 in resting cells, at low Ca(2+) concentration. Annexin 2 might be associated with membrane domains enriched in phosphatidylserine and cholesterol.