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P Fort

Publications and source records attributed to P Fort.

At least 37 records · Page 2Linked to original sources
PubMed2000

TrioGEF1 controls Rac- and Cdc42-dependent cell structures through the direct activation of rhoG.

Rho GTPases regulate the morphology of cells stimulated by extracellular ligands. Their activation is controlled by guanine exchange factors (GEF) that catalyze their binding to GTP. The multidomain Trio protein represents an emerging class of &Rgr; regulators that contain two GEF domains of distinct specificities. We report here the characterization of Rho signaling pathways activated by the N-terminal GEF domain of Trio (TrioD1). In fibroblasts, TrioD1 triggers the formation of particular cell structures, similar to those elicited by RhoG, a GTPase known to activate both Rac1 and Cdc42Hs. In addition, the activity of TrioD1 requires the microtubule network and relocalizes RhoG at the active sites of the plasma membrane. Using a classical in vitro exchange assay, TrioD1 displays a higher GEF activity on RhoG than on Rac1. In fibroblasts, expression of dominant negative RhoG mutants totally abolished TrioD1 signaling, whereas dominant negative Rac1 and Cdc42Hs only led to partial and complementary inhibitions. Finally, expression of a Rho Binding Domain that specifically binds RhoG(GTP) led to the complete abolition of TrioD1 signaling, which strongly supports Rac1 not being activated by TrioD1 in vivo. These data demonstrate that Trio controls a signaling cascade that activates RhoG, which in turn activates Rac1 and Cdc42Hs.

Actins↗
PubMed2000

Rho and Rac exert antagonistic functions on spreading of macrophage-derived multinucleated cells and are not required for actin fiber formation.

Multinucleated giant cells (MNGC) derived from avian blood monocytes present, like osteoclasts, an unusual cytoskeletal organization characterized by (1) cortical rings of actin filaments, (2) unique adhesion structures called podosomes and (3) vinculin containing focal complexes which are not visibly connected to F-actin structures. The Rho family of small GTPases plays an essential role in the regulation and organization of cellular cytoskeletal structures including F-actin and vinculin associated structures. Using bacterial toxins such as modified exoenzyme C3 (C3B) and toxin B or overexpression of constitutively active Rac and Rho proteins fused to the green fluorescent protein (GFP), we show that Rac and Rho play antagonistic roles in regulating the morphology of osteoclast-like cells. Inhibition of Rho by C3B triggered MNGC spreading whereas activated Rho promoted cell retraction. However, inhibition or activation of Rho led to complete disorganization of fibrillar actin structures, including podosomes. Toxin B inhibition of Rho, Rac and Cdc42 induced a time dependent F-actin and vinculin reorganization. Initially, actin fibers with associated adhesion plaques formed and disappeared subsequently. Finally, only small focal complexes remained at the MNGC periphery before retracting. At the time when actin fibers formed, we observed that Rac was already inhibited by toxin B. By combining C3B treatment and overexpression of a dominant negative form of Rac (N17Rac), we show that the formation of these focal adhesion and actin fiber structures required neither Rho nor Rac activity. Moreover, our results show that podosomes are extremely unstable structures since any modifications of Rho or Rac activity resulted in their dissociation.

3T3 Cells↗
PubMed1999

Origins of the glycinergic inputs to the rat locus coeruleus and dorsal raphe nuclei: a study combining retrograde tracing with glycine immunohistochemistry.

The amino acid glycine is a major inhibitory neurotransmitter in the brainstem and is likely involved in the tonic inhibition of the monoaminergic neurons during all sleep-waking stages. In order to determine the neurons at the origin of the glycinergic innervation of the two principal monoaminergic nuclei, the locus coeruleus and the dorsal raphe of the rat, we applied a double-labelling technique, combining retrograde transport of cholera-toxin B subunit with glycine immunohistochemistry. Using this technique, we found that the locus coeruleus and dorsal raphe nuclei receive a common glycinergic innervation from the ventral and ventrolateral periaqueductal grey, including the adjacent deep mesencephalic reticular nucleus. Small additional glycinergic inputs to these nuclei originated from the lateral paragigantocellular nucleus and the rostral ventromedial medullary reticular formation. The potential role of these glycinergic inputs in the control of the excitability of the monoaminergic neurons of the locus coeruleus and dorsal raphe nuclei is discussed.

Animals↗
PubMed1999

Rho family GTPases control entry of Shigella flexneri into epithelial cells but not intracellular motility.

Shigella flexneri, an invasive bacterial pathogen, promotes formation of two cytoskeletal structures: the entry focus that mediates bacterial uptake into epithelial cells and the actin-comet tail that enables the bacteria to spread intracellularly. During the entry step, secretion of bacterial invasins causes a massive burst of subcortical actin polymerization leading the formation of localised membrane projections. Fusion of these membrane ruffles leads to bacterial internalization. Inside the cytoplasm, polar expression of the IcsA protein on the bacterial surface allows polymerization of actin filaments and their organization into an actin-comet tail leading to bacterial spread. The Rho family of small GTPases plays an essential role in the organization and regulation of cellular cytoskeletal structures (i.e. filopodia, lamellipodia, adherence plaques and intercellular junctions). We show here that induction of Shigella entry foci is controlled by the Cdc42, Rac and Rho GTPases, but not by RhoG. In contrast, actin-driven intracellular motility of Shigella does not require Rho GTPases. Therefore, Shigella appears to manipulate the epithelial cell cytoskeleton both by Rho GTPase-dependent and -independent processes.

Actins↗
PubMed1998

A presumptive developmental role for a sea urchin cyclin B splice variant.

We show that a splice variant-derived cyclin B is produced in sea urchin oocytes and embryos. This splice variant protein lacks highly conserved sequences in the COOH terminus of the protein. It is found strikingly abundant in growing oocytes and cells committed to differentiation during embryogenesis. Cyclin B splice variant (CBsv) protein associates weakly in the cell with Xenopus cdc2 and with budding yeast CDC28p. In contrast to classical cyclin B, CBsv very poorly complements a triple CLN deletion in budding yeast, and its microinjection prevents an initial step in MPF activation, leading to an important delay in oocyte meiosis reinitiation. CBsv microinjection in fertilized eggs induces cell cycle delay and abnormal development. We assume that CBsv is produced in growing oocytes to keep them in prophase, and during embryogenesis to slow down cell cycle in cells that will be committed to differentiation.

Alternative Splicing↗
PubMed1998

Pharmacological characterization and differentiation of non-cholinergic nucleus basalis neurons in vitro.

Using intracellular recordings in guinea pig brain slices, the pharmacology of electrophysiologically identified and immunohistochemically confirmed non-cholinergic nucleus basalis neurons was studied to determine their response to the major neurotransmitters of the subcortical afferents to this region. The cells were differentiated into three types: Type A cells (approximately 44%) were depolarized by noradrenaline (NA) and muscarine, Type B cells (approximately 23%) were depolarized by NA but hyperpolarized by muscarine, and Type C cells (approximately 15%) were hyperpolarized by both agonists. These cell types were also differentially responsive to serotonin (hyperpolarizing B, C) and histamine (depolarizing A, B). Accordingly, the non-cholinergic neurons share certain discharge properties but appear nonetheless to comprise distinct types which respond differentially to the major modulatory neurotransmitters and thus play potentially different roles in cortical modulation across the sleep-wake cycle.

Animals↗
PubMed1998

GABAergic input to cholinergic nucleus basalis neurons.

The potential influence of GABAergic input to cholinergic basalis neurons was studied in guinea-pig basal forebrain slices. GABA and its agonists were applied to electrophysiologically-identified cholinergic neurons, of which some were labelled with biocytin and confirmed to be choline acetyltransferase-immunoreactive. Immunohistochemistry for glutamate decarboxylase was also performed in some slices and revealed GABAergic varicosities in the vicinity of the biocytin-filled soma and dendrites of electrophysiologically-identified cholinergic cells. From rest (average - 63 mV), the cholinergic cells were depolarized by GABA. The depolarization was associated with a decrease in membrane resistance and diminution in firing. The effect was mimicked by muscimol, the specific agonist for GABA(A) receptors, and not by baclofen, the specific agonist for GABA(B) receptors, which had no discernible effect. The GABA- and muscimol-evoked depolarization and decrease in resistance were found to be postsynaptic since they persisted in the presence of solutions containing either high Mg2+/low Ca2+ or tetrodotoxin. They were confirmed as being mediated by a GABA(A) receptor, since they were antagonized by bicuculline. The reversal potential for the muscimol effect was estimated to be approximately -45 mV, which was -15 mV above the resting membrane potential. Finally, in some cholinergic cells, spontaneous subthreshold depolarizing synaptic potentials (average 5 mV in amplitude), which were rarely associated with action potentials, were recorded and found to persist in the presence of glutamate receptor antagonists but to be eliminated by bicuculline. These results suggest that GABAergic input may be depolarizing, yet predominantly inhibitory to cholinergic basalis neurons.

2-Amino-5-phosphonovalerate↗
PubMed1998

Electrophysiological evidence that noradrenergic neurons of the rat locus coeruleus are tonically inhibited by GABA during sleep.

It is well known that noradrenergic locus coeruleus (LC) neurons decrease their activity during slow wave sleep (SWS) and are virtually quiescent during paradoxical sleep (PS). It has been proposed that a GABAergic input could be directly responsible for this sleep-dependent neuronal inactivation. To test this hypothesis, we used a new method combining polygraphic recordings, microiontophoresis and single-unit extracellular recordings in unanaesthetized head-restrained rats. We found that iontophoretic application of bicuculline, a specific GABA(A)-receptor antagonist, during PS and SWS restore a tonic firing in the LC noradrenergic neurons. We further observed that the application of bicuculline during wakefulness (W) induced an increase of the discharge rate. Of particular importance for the interpretation of these results, using the microdialysis technique, Nitz and Siegel (Neuroscience, 1997; 78: 795) recently found an increase of the GABA release in the cat LC during SWS and PS as compared with waking values. Based on these and our results, we therefore propose that during W, the LC cells are under a GABAergic inhibitory tone which progressively increases at the entrance and during SWS and PS and is responsible for the inactivation of these neurons during these states.

Animals↗
PubMed1998

RhoG GTPase controls a pathway that independently activates Rac1 and Cdc42Hs.

RhoG is a member of the Rho family of GTPases that shares 72% and 62% sequence identity with Rac1 and Cdc42Hs, respectively. We have expressed mutant RhoG proteins fused to the green fluorescent protein and analyzed subsequent changes in cell surface morphology and modifications of cytoskeletal structures. In rat and mouse fibroblasts, green fluorescent protein chimera and endogenous RhoG proteins colocalize according to a tubular cytoplasmic pattern, with perinuclear accumulation and local concentration at the plasma membrane. Constitutively active RhoG proteins produce morphological and cytoskeletal changes similar to those elicited by a simultaneous activation of Rac1 and Cdc42Hs, i.e., the formation of ruffles, lamellipodia, filopodia, and partial loss of stress fibers. In addition, RhoG and Cdc42Hs promote the formation of microvilli at the cell apical membrane. RhoG-dependent events are not mediated through a direct interaction with Rac1 and Cdc42Hs targets such as PAK-1, POR1, or WASP proteins but require endogenous Rac1 and Cdc42Hs activities: coexpression of a dominant negative Rac1 impairs membrane ruffling and lamellipodia but not filopodia or microvilli formation. Conversely, coexpression of a dominant negative Cdc42Hs only blocks microvilli and filopodia, but not membrane ruffling and lamellipodia. Microtubule depolymerization upon nocodazole treatment leads to a loss of RhoG protein from the cell periphery associated with a reversal of the RhoG phenotype, whereas PDGF or bradykinin stimulation of nocodazole-treated cells could still promote Rac1- and Cdc42Hs-dependent cytoskeletal reorganization. Therefore, our data demonstrate that RhoG controls a pathway that requires the microtubule network and activates Rac1 and Cdc42Hs independently of their growth factor signaling pathways.

3T3 Cells↗
PubMed1998

Anatomical demonstration of a medullary enkephalinergic pathway potentially implicated in the oro-facial muscle atonia of paradoxical sleep in the cat.

The present study was aimed to compare in detail the distribution within the rostral ventromedial medulla of Methionin-Enkephalin-immunoreactive neurons with efferent projections to the facial or trigeminal motor nuclei, using a double immunostaining technique in colchicine-treated cats. Following cholera toxin B subunit injections in the facial or trigeminal motor nuclei, we found that respectively 55% and 65% of the medium to large-sized retrogradely labeled cells in the lateral part of the nucleus reticularis magnocellularis were Methionin-Enkephalin-positive. For both motor nuclei, the double-labeled neurons had similar morphology and size and were located exactly in the same area. They could therefore belong to the same population of reticular enkephalinergic neurons. Based on these and previous anatomical and electrophysiological data, we propose that these enkephalin-containing neurons could participate in the hyperpolarization of brainstem and spinal somatic motoneurons during paradoxical sleep.

Animals↗
PubMed1997

Structure and chromosomal assignment to 22q12 and 17qter of the ras-related Rac2 and Rac3 human genes.

Members of the Rho/Rac/Cdc42Hs family of GTPases have been shown to participate in many aspects of the signaling of cell growth and differentiation. Although the biochemical properties of these GTPases have been extensively studied, very little is known about the structure of the corresponding genes. To gain insight on the evolution of the Rho family, we were interested in studying the genomic structure of several members. We report here the structure and the localization to 22q12 of the human Rac2 gene, as well as the localization to 17qter of Rac3, a new member closely related to Rac1 and Rac2. Unlike the structure of its closest relative ARH-G gene, which contains a single intron, Rac2 is made of at least 7 exons, spanning over 18 kb of DNA. Comparison of gene structure and exonic borders suggests that the emergence of the whole superfamily took place early during evolution.

Amino Acid Sequence↗
PubMed1997

The small GTPases Cdc42Hs, Rac1 and RhoG delineate Raf-independent pathways that cooperate to transform NIH3T3 cells.

BACKGROUND: Ras-mediated transformation of mammalian cells has been shown to activate multiple signalling pathways, including those involving mitogen-activated protein kinases and the small GTPase Rho. Members of the Rho family affect cell morphology by controlling the formation of actin-dependent structures: specifically, filopodia are induced by Cdc42Hs, lamellipodia and ruffles by Rac, and stress fibers by RhoA. In addition, Rho GTPases are involved in progression through the G1 phase of the cell cycle, and Rac1 and RhoA have recently been directly implicated in the morphogenic and mitogenic responses to transformation by oncogenic Ras. In order to examine the cross-talk between Ras and Rho proteins, we investigated the effects on focus-forming activity and cell growth of the Rho-family members Cdc42Hs, Rac1 and RhoG by expressing constitutively active or dominant-negative forms in NIH3T3 cells. RESULTS: Expression of Rac1 or RhoG modulated the saturation density to which the cells grew, probably by affecting the level of contact inhibition. Although all three GTPases were required for cell transformation mediated by Ras but not by constitutively active Raf, the selective activation of each GTPase was not sufficient to induce the formation of foci. The coordinated activation of Cdc42Hs, RhoG and Rac1, however, elicited a high focus-forming activity, independent of the mitogen-activated ERK and JNK protein kinase pathways. CONCLUSIONS: Ras-mediated transformation induces extensive changes in cell morphology which require the activity of members of the Rho family of GTPases. Our data show that the pattern of coordinated Rho family activation that elicits a focus-forming activity in NIH3T3 cells is distinct from the regulatory cascade that has been proposed for the control of actin-dependent structures in Swiss 3T3 cells.

3T3 Cells↗
PubMed1997

Structure of the human ARHG locus encoding the Rho/Rac-like RhoG GTPase.

Members of the Rho/Rac/Cdc42Hs family of GTPases have been shown to participate in many aspects of the signaling of cell growth and differentiation. Although the biochemical properties of these GTPases have been extensively studied, very little is known about their gene structure and regulation. RhoG, a member related to Rac and Cdc42Hs, is activated at the transcriptional level in the mid-G1 phase of stimulated fibroblasts. As a first step toward the characterization of the regulatory elements involved in serum-regulated expression, we isolated and determined the structure of the corresponding human locus (ARHG, localized in 11p15.4-p15.5). This is the first gene structure of a member of the Rho/Rac/Cdc42Hs family. At variance with Ras and Rab3A genes, ARHG contains a single intron larger than 20 kb that splits a 62-nt-long 5' noncoding first exon from the rest of the mRNA. The sequences upstream of the cap sites exhibit transcriptional activity. They are G/C-rich and devoid of TATA or CAAT boxes, as found for many housekeeping genes, including Ras genes.

Animals↗
PubMed1997

Expression and human chromosomal localization to 17q25 of the growth-regulated gene encoding the mitochondrial ribosomal protein MRPL12.

Mitochondrial activity requires the expression of nuclear genes, whose products are part of multiproteic complexes leading to ATP production and delivery. We recently characterized a growth-activated mRNA encoding the human mitochondrial ribosomal MRPL12 protein, which is thought to act as a translational regulator of mitochondrial mRNAs. We show here that MRPL12 mRNA expression is enhanced in growth-stimulated cells as a result of transcriptional activation, a feature lost in transformed cell lines. MRPL12 mRNA is highly expressed in the colon, in which a reduction in mitochondrial activity was shown to be associated with tumor formation. The human MRPL12 protein is encoded by a unique gene located on chromosome 17 (q25-qter). As no predisposition to colon cancer linked to this chromosomal region was hitherto reported, the MRPL12 gene might be involved in the process of differentiation of colonic epithelial cells.

Animals↗
PubMed1997

A simple luciferase assay for signal transduction activity detection of epidermal growth factor displayed on phage.

Studies on receptor-ligand interactions are important for the design of agonists or antagonists of natural ligands. We developed a luciferase reporter assay to screen epidermal growth factor receptor (EGFR) binding molecules rapidly for their ability to stimulate or inhibit signal transduction. Human EGF displayed on fd filamentous phage presented an activity similar to soluble EGF when tested for binding to the EGFR, for induction of cell cycle progression or in the luciferase assay. Two libraries of human EGF variants displayed on phage were constructed in which the aspartic acid residue at position 46 or the arginine residue at position 41 were randomised. EGF mutants displayed on phage were screened in parallel for binding to the EGFR using an ELISA assay and for transducing activity using the luciferase assay. Regarding the 46 position, most of the mutants retained the ability to bind the EGFR and their transducing activity corresponded perfectly with their binding. For the more crucial 41 position, only the wild-type EGF was able to bind the EGFR. Our approach allowed a simple determination of crucial positions and paved the way for identification of agonists with altered transduction activity.

3T3 Cells↗
PubMed1997

Modulation of cholinergic nucleus basalis neurons by acetylcholine and N-methyl-D-aspartate.

Known to exert an important modulatory influence on the cerebral cortex, the cholinergic neurons of the basal forebrain are modulated in turn by neurotransmitters which may include acetylcholine released from processes of brainstem or forebrain neurons. In the present study, we examined the effect of carbachol, a non-specific cholinergic agonist, either alone or in the presence of N-methyl-D-aspartate upon electrophysiologically identified cholinergic basalis neurons in guinea-pig basal forebrain slices. Carbachol produced a direct postsynaptic hyperpolarization, accompanied by a decrease in membrane resistance. Muscarine could mimic this hyperpolarizing effect, whereas nicotine produced a direct postsynaptic membrane depolarization. The interaction of carbachol with N-methyl-D-aspartate was subsequently tested since, in a prior study, N-methyl-D-aspartate was shown to induce rhythmic bursting in cholinergic cells when they were hyperpolarized by continuous injection of outward current. Applied simultaneously with N-methyl-D-aspartate in the absence of current injection, carbachol was also found to promote rhythmic bursting in half of the cells tested. Since the bursts under these conditions were markedly longer in duration than those observed in the presence of N-methyl-D-aspartate alone, it was hypothesized that carbachol might have another action, in addition to the membrane hyperpolarization. Using dissociated cells, it was found that brief applications of carbachol could indeed diminish the slow afterhyperpolarizations that follow single spikes, short bursts or long trains of action potentials in cholinergic basalis neurons. These results indicate that, through its dual ability to hyperpolarize cholinergic neurons and to reduce their afterhyperpolarizations, acetylcholine can promote the occurrence of rhythmic bursting in the presence of N-methyl-D-aspartate. Accordingly, whether derived from brainstem or local sources, acetylcholine may facilitate rhythmic discharge in cholinergic basalis neurons which could in turn impose a rhythmic modulation upon cortical activity during particular states across the sleep-waking cycle.

Acetylcholine↗