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First published online March 2, 2004
doi: 10.1242/10.1242/jcs.00927

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Depolarization-induced translocation of the RNA-binding protein Sam68 to the dendrites of hippocampal neurons

Naïla Ben Fredj¹, Julien Grange¹, Rémy Sadoul¹, Stéphane Richard², Yves Goldberg^1,3,*, and Véronique Boyer^1,*,

¹ Neurodégénérescence et Plasticité, INSERM EMI 01-08, Institut National de la Santé et de la Recherche Médicale, Pavillon de Neurologie, Centre Hospitalier Universitaire, 38043 Grenoble, France
² Departments of Oncology and Medicine, Lady Davis Institute for Medical Research and McGill University, Montreal, Quebec H3T 1E2, Canada
³ Département de Réponse et Dynamique Cellulaires, Commissariat à l'Energie Atomique (CEA), 38054 Grenoble, France

View larger version (67K): [in a new window]   Fig. 1. Nucleo-somatodendritic translocation of Sam68 in depolarized rat hippocampal neurons. (A) Immunostaining of Sam68 using the AD1 rabbit anti-serum in neurons at 9 DIV cultured at 5 mM KCl showing a nuclear labeling (left) or at 25 mM KCl showing a perinuclear staining or a somatodendritic staining (right, see arrows). (B) Confocal image of a same neuron cultured at 25 mM KCl labeled with the anti-Sam68 AD1. Sam68 was concentrated in clusters (arrowheads, red fluorescence, upper panel) and MAP2 antibodies (green fluorescence, lower panel).

View larger version (42K): [in a new window]   Fig. 2. Distribution of Sam68 in GFP-Sam68-transfected hippocampal neurons. (A) Quantification and representative images of the subcellular localization of GFP-Sam68 in nuclei (N), nuclei + soma (N+S) and in nuclei + soma + dendrites (N+S+D). Error bars represent s.d. (n=3, where 200-300 transfected cells were counted). (B) Nucleo-somatodendritic expression of the N-terminal deleted GFP-Sam68 mutant transfected neuron. (C) GFP-Sam68-transfected neuron where individual granules moving into dendrites of living hippocampal neurons could be visualized. Transfected neurons were incubated in control medium as described in Materials and Methods and individual motilities of green fluorescent granules were observed by time-lapse video microscopy for 8 hours each 20 minutes. Images taken at t=0 and t=480 minutes are shown. Arrows label moving granules, whereas the arrowhead labels a stationary granule.

View larger version (61K): [in a new window]   Fig. 3. Association of GFP-Sam68 granules with RNA. (A) Confocal images of dendritic localization of GFP-Sam68 in hippocampal neurons showing fluorescent granules (green fluorescence, GFP-Sam68) that colocalize in three regions (yellow fluorescence, merged image) with RNA labeling by ethidium bromide (red fluorescence). Percentage of colocalization of GFP-Sam68 and ethidium bromide. Error bar represents s.d. (n=100). (B) RNAse I treatment of the EtBr labeling of neurons. (C) DNAse I treatment of the EtBr labeling of neurons. (D) Binding of immunoprecipitated Sam68 from adult rat cortex with ß-actin mRNA. RT-PCR with specific ß-actin primers was carried out with AD1 anti-Sam68 antibody immunoprecipitation (lane 1), with preimmune IgG immunoprecipitation (lane 2), with total extract (lane 3) or with no input (lane 4).

View larger version (34K): [in a new window]   Fig. 4. Translocation of GFP-Sam68 from the nucleus to the somato-dendritic compartment in depolarized hippocampal neurons. (A) GFP-Sam68CREs-transfected (left panel) or GFP-SC35CREs-transfected (right panel) neurons were incubated with 5 mM KCl (white) or with 25 mM KCl (hatched or stippled) for 5 hours. Quantification of the subcellular localization of GFP-Sam68 was then reported as a percentage of transfected neurons showing nuclei (N), nuclei + soma (N+S) and nuclei + soma + dendrites (N+S+D) localization (n=3, where 200-300 transfected cells were counted). (B) Patterns of expression of Sam68 in GFP-Sam68CREs- or GFP-SC35CREs-transfected neurons depolarized (25 mM KCl) or not (5 mM KCl) for 5 hours. (C) Tyrosine-phosphorylation pattern of neurons depolarized (25 mM KCl) or not (5 mM KCl) for 5 hours.

View larger version (52K): [in a new window]   Fig. 5. Dendritic microtubule-dependent transport of Sam68 during depolarization. (A) Effect in percent of a microtubule-disrupting drug vincristine (5 µM) in depolarized neurons. GFP-Sam68CREs-transfected neurons were incubated with 25 mM KCl for 5 hours in the absence (hatched) or presence (stippled) of vincristine. Error bars represent s.d. (n=3, where 200-300 transfected cells were counted). (B) Images of the -tubulin immunostaining in depolarized neurons in the absence or presence of vincristine. (C) Images of the GFP-Sam68 expression pattern in depolarized neurons in the absence or presence of vincristine.

View larger version (46K): [in a new window]   Fig. 6. Inhibition of Sam68 translocation during depolarization. (A) Neurons were depolarized with 25 mM KCl for 5 hours in the absence (–) or presence of nimodipine (5 µM) or leptomycin B (LMB, 60 ng/ml) to block calcium entry through voltage-sensitive calcium channels or CRM-1 nuclear export, respectively. s.d. with n=3, where 200-300 transfected cells were counted (percentages of neurons with nuclear and somatic Sam68 expression are not shown). (B) Images of neurons transfected with GFP-Rev-NES or GFP-Sam68CREs treated or not with LMB (60 ng/ml) for 5 hours.