Técnicas de microscopía óptica

Durante los últimos años, las técnicas de microscopía óptica y sus aplicaciones han experimentado un auge sin precedentes. Más concretamente, la difusión de sistemas de microscopía confocal durante los 90 ha empujado con fuerza el desarrollo de la microscopía de fluorescencia y abierto nuevos horizontes de investigación. Este fenómeno ha venido propiciado por varios motivos. Por una parte, los progresos en la tecnología de detectores fotosensibles, tanto chips CCD (charge-coupled devices) como fotomultiplicadores (PMT), cada vez de mayor resolución y sensibilidad.

Role of phosphorylated MAPIB in neuritogenesis

The distribution of microtubule-associated protein lB (MAPlB) phosphorylated by either proline-directed protein kinase (PDPK) or casein kinase II (CK II) in neuroblastoma cells and hippocampal neurons has been studied by immunofluorescence using specific antibodies to distinct phosphorylation-sensitive epitopes. A proximo-distal gradient of increasing PDPK-catalyzed phosphorylation of MAPlB is superimposed on a proximo distal gradient of decreasing CK II-catalyzed MAPlB phosphorylation within growing axon-like neurites. Additionally, CK II-phosphorylated MAPlB is present in cell bodies and dendrites where no PDPK-phosphorylated MAPlB is observed. These results suggest distinct roles for both types of modifications of MAPlB in developing neurons.Peer reviewe

Neurogranin in the development of the rat telencephalon

We have used a novel antibody to map the distribution of the protein kinase C substrate protein RC3/neurogranin during the development of the rat telencephalon. Neurogranin appearance in the rat brain is biphasic: it shows an early stage of anatomically restricted, low-intensity expression, and a juvenile stage of anatomically widespread, high-intensity expression. Most of the structures that express neurogranin during development conserve it in the adult stage. Neurogranin expression starts on embryonic day 18 in two different sites-the amygdalar primordium and in the piriform cortex-and is confined to these structures until the first postnatal day (P1). On P1, neurogranin expression increases dramatically in intensity, and appears in the olfactory cortex, isocortex, subiculum and hippocampus. In the striatum, expression starts on P1 and extends to the caudoputamen and parts of the globus pallidus and septum. Particularly complex patterns of labelling can be seen in the amygdala and cerebral cortex. Cortical layers showing early expression are the presumptive layers 4 and 5 in the somatosensory cortex, and layers 2 and 5 in the anterior cingulate and agranular insular cortices. Immunoreactivity is found mostly in cell bodies during the early and juvenile stages, but by the end of the first postnatal week it starts being more apparent in the neuropil. This phenomenon probably reflects the intracellular translocation of neurogranin to distal parts of the dendrites and dendritic spines. This process culminates by the end of the second postnatal week, when the adult pattern is reached. According to the timing and anatomy of its distribution, expression of neurogranin seems to be independently regulated in each telencephalic region by specific signalling mechanisms. It is proposed, on this basis, that neurogranin could be implicated in neuronal differentiation and synaptogenesis during telencephalic development.Peer Reviewe

Kir2.1-Nav1.5 channel complexes are differently regulated than Kir2.1 and Nav1.5 channels alone

Cardiac Kir2.1 and Nav1.5 channels generate the inward rectifier K+ (I) and the Na+ (I) currents, respectively. There is a mutual interplay between the ventricular I and I densities, because Nav1.5 and Kir2.1 channels exhibit positive reciprocal modulation. Here we compared some of the biological properties of Nav1.5 and Kir2.1 channels when they are expressed together or separately to get further insights regarding their putative interaction. First we demonstrated by proximity ligation assays (PLAs) that in the membrane of ventricular myocytes Nav1.5 and Kir2.1 proteins are in close proximity to each other (< 40 nm apart). Furthermore, intracellular dialysis with anti-Nav1.5 and anti-Kir2.1 antibodies suggested that these channels form complexes. Patch-clamp experiments in heterologous transfection systems demonstrated that the inhibition of the Ca/calmodulin-dependent protein kinase II (CaMKII) decreased the I and the I generated by Nav1.5 and Kir2.1 channels when they were coexpressed, but not the I generated by Kir2.1 channels alone, suggesting that complexes, but not Kir2.1 channels, are a substrate of CaMKII. Furthermore, inhibition of CaMKII precluded the interaction between Nav1.5 and Kir2.1 channels. Inhibition of 14-3-3 proteins did not modify the I and I densities generated by each channel separately, whereas it decreased the I and I generated when they were coexpressed. However, inhibition of 14-3-3 proteins did not abolish the Nav1.5-Kir2.1 interaction. Inhibition of dynamin-dependent endocytosis reduced the internalization of Kir2.1 but not of Nav1.5 or Kir2.1-Nav1.5 complexes. Inhibition of cytoskeleton-dependent vesicular trafficking via the dynein/dynactin motor increased the I, but reduced the I, thus suggesting that the dynein/dynactin motor is preferentially involved in the backward and forward traffic of Kir2.1 and Nav1.5, respectively. Conversely, the dynein/dynactin motor participated in the forward movement of Kir2.1-Nav1.5 complexes. Ubiquitination by Nedd4-2 ubiquitin-protein ligase promoted the Nav1.5 degradation by the proteasome, but not that of Kir2.1 channels. Importantly, the Kir2.1-Nav1.5 complexes were degraded following this route as demonstrated by the overexpression of Nedd4-2 and the inhibition of the proteasome with MG132. These results suggested that Kir2.1 and Nav1.5 channels closely interact with each other leading to the formation of a pool of complexed channels whose biology is similar to that of the Nav1.5 channels.Fondos Europeos de Desarrollo Regional; Ministerio de Economía y CompetitividadPeer Reviewe

Microtubule-associated protein 2 phosphorylation is decreased in the human epileptic temporal lobe cortex

Microtubule-associated protein 2 (MAP2) is an abundant component of the neuronal cytoskeleton whose function is related to the outgrowth and stability of neuronal processes, to synaptic plasticity and neuronal cell death. We have sought to study whether abnormal patterns of neuronal activity which are characteristic of epileptic patients are associated to alterations of MAP2 phosphorylation. An antibody (305) that selectively recognizes a phosphorylated epitope in a proline-rich region of the MAP2 molecule has been used to analyze neocortical biopsy samples from temporal lobe epileptic patients, whose electrocorticogram activity had been previously monitored. Immunoblot analysis showed that samples with greater spiking activity displayed significantly diminished MAP2 phosphorylation. Immunocytochemical analysis revealed the occurrence of discrete areas in the neocortex with highly decreased or no immunostaining for antibody 305, which showed a clear, although non-significant, tendency to appear more frequently in areas with greater spiking activity. To further support an association between epileptiform activity and MAP2 dephosphorylation an experimental model of epileptiform activity in cultures of rat hippocampal neurons was used. Neurons were cultured during 15 days in the presence of kynurenic acid, an antagonist of glutamate receptors. At this time, kynurenic acid was removed from the culture medium and neurons developed seizure-like activity. Using antibody 305, we found a decrease of MAP2 phosphorylation that was already visible after 15 min of kynurenic acid withdrawal. We therefore propose that MAP2 phosphorylation is decreased in the neocortex of epileptic patients and that this decrease is a likely consequence of seizure activity. Also, MAP2 dephosphorylation may lead to alterations of the neuronal cytoskeleton and eventually to neuronal damage and loss, which is typical of epileptic patients.Financial support for this study came from DGCYT Grants SAF96-0032, PM98-0009 and PM99-0105, ‘Comunidad de Madrid’ Grants 08.5/0020/1999 and 0.8.5/0023/1999, a grant from ‘Fundación La Caixa’, a Gobierno Vasco grant (J.I.A.) and a post-doctoral grant from ‘Comunidad de Madrid’ (C.S.). We thank ‘Fundación Ramón Areces’ for institutional support.Peer reviewe

Mechanisms of endoplasmic reticulum export of the glycine transporter GLYT1

International audienceThe glycine transporter GLYT1 regulates both glycinergic and glutamatergic neurotransmission by controlling the reuptake of glycine at synapses. Trafficking to the cell surface of GLYT1 is critical for its function. In this article, by using mutational analysis of the GLYT1 C-terminal domain we identified the evolutionary conserved motif R 575}L 576}(X8)D 585} as being necessary for endoplasmic reticulum (ER) export. This is probably due to its capacity to bind Sec24D, a component of the COPII complex. This ER export motif was active when introduced into the related GLYT2 transporter but not in the unrelated VSVG protein. GLYT1 protein in which this motif was mutated was not transported to the plasma membrane, although this effect was rescued by co-expressing these mutants with wild-type GLYT1. This behavior suggests that GLYT1 might form oligomers along the trafficking pathway. Cross-linking assays performed in rat brain synaptosomes, and Fluorescence Resonance Energy Transfer (FRET) microscopy in living cells confirmed the existence of GLYT1 oligomers. In summary, we have identified a motif involved in the ER exit of GLYT1 and in analyzing the influence of this motif, we have found evidence that oligomerization is important for the trafficking of GLYT1 to the cell surface. Because this motif is conserved in the NSS family, it is possible that this finding could be extrapolated to other related transporters

Visualization of Phosphatidic Acid Fluctuations in the Plasma Membrane of Living Cells

<div><p>We developed genetically-encoded fluorescent sensors based on Förster Resonance Energy Transfer to monitor phosphatidic acid (PA) fluctuations in the plasma membrane using Spo20 as PA-binding motif. Basal PA levels and phospholipase D activity varied in different cell types. In addition, stimuli that activate PA phosphatases, leading to lower PA levels, increased lamellipodia and filopodia formation. Lower PA levels were observed in the leading edge than in the trailing edge of migrating HeLa cells. In MSC80 and OLN93 cells, which are stable cell lines derived from Schwann cells and oligodendrocytes, respectively, a higher ratio of diacylglycerol to PA levels was demonstrated in the membrane processes involved in myelination, compared to the cell body. We propose that the PA sensors reported here are valuable tools to unveil the role of PA in a variety of intracellular signaling pathways.</p></div

Activity-dependent translocation of neurogranin to neuronal nuclei

International audienceLong-term changes of synaptic plasticity depend on protein synthesis and transcription. Neurogranin (Ng) is a small protein concentrated at dendrites and spines of forebrain neurons, involved in synaptic plasticity through the regulation of calmodulin (CaM) mediated signalling. Ng presents a central IQ motif that mediates its binding to CaM and phosphatidic acid (PA) and that can be phosphorylated by protein kinase C (PKC). Here, we report that Ng displays a strong nuclear localization when expressed in cell lines and hippocampal neurons, either alone or fused to green fluorescent protein (GFP-Ng). Further, using subcellular fractionation and immunocytochemical techniques, we were able to localize endogenous Ng in the nuclei of rat forebrain neurons. Nuclear localization of Ng depends on its IQ motif and is reduced by binding to cytoplasmic CaM. Also, PKC stimulation induces a transient nuclear translocation of Ng in acute hippocampal slices. A similar translocation is observed in neurons of the cerebral cortex and hippocampus after the induction of generalized seizures in adult rats. In summary, the data presented here show that a fraction of rat brain Ng is localized in the neuronal nuclei and that synaptic activity regulates its translocation from the cytoplasm. The possible involvement of Ng in the regulation of intranuclear Ca2+/CaM dependent signalling and gene expression is discussed

Response of pmPAS to liposomes containing dioleoylphosphatidic acid or oleic acid.

<p>(<b>A</b>) Fluorescence intensity (Venus channel) and ECFP/FRET ratio images of a HeLa cell expressing pmPAS challenged with liposomes containing dioleoylphosphatidic acid (lipoDOPA, 200 µM). (<b>B</b>) Time course of normalized ECFP/FRET of the cell shown in (A). The time course and magnified images refer to the area indicated by a box in the gray image. (<b>C</b>) ECFP/FRET ratio images of a HeLa cell expressing pmPAS at the indicated time (min) before or after addition of liposomes containing oleic acid (lipoOA, 500 µM). (<b>D</b>) Time course of normalized ECFP/FRET of cells challenged with lipoOA (as in (C)) (the cell average of 4 cells is shown), or incubated with phosphatidylcholine liposomes (lipoPC) (<i>n</i> = 6, 3 independent experiments). Error bars indicate the mean±SEM. Scale bars represent 20 µm and the ECFP/FRET images were coded according to the indicated pseudocolor scale.</p

Variability of plasma membrane PA levels in various cancer cell lines.

<p>HeLa, HCT116 and HT29 cells were transiently transfected with pmPAS. (<b>A</b>) Basal ECFP/FRET ratios on regions of interest on the plasma membrane were determined (HeLa <i>n</i> = 45, HT29 <i>n</i> = 48, HCT116 <i>n</i> = 53, ***<i>p</i><0.0001 using one-way ANOVA). (<b>B</b>) Time course of ECFP/FRET values of cells in response to PMA (100 nM) (HT29: <i>n</i> = 7, 3 independent experiments; HCT116: <i>n</i> = 6, 3 independent experiments; HeLa: <i>n</i> = 9, 4 independent experiments) (<b>C</b>) Time course of normalized ECFP/FRET values of cells in response to FIPI (1 µM) (HT29: <i>n</i> = 6, 4 independent experiments; HCT116: <i>n</i> = 8, 4 independent experiments; HeLa: <i>n</i> = 9, 4 independent experiments), respectively. Error bars indicate the mean±SEM.</p