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First published online July 12, 2005
doi: 10.1242/10.1242/jcs.02446

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Nuclear inositol 1,4,5-trisphosphate receptors regulate local Ca²⁺ transients and modulate cAMP response element binding protein phosphorylation

¹ Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago 7, Chile
² Institut Fédératif de Neurobiologie Alfred Fessard, Laboratoire de Neurobiologie Cellulaire et Moléculaire, UPR 9040 CNRS, 91198 Gif-sur-Yvette CEDEX, France
³ Laboratorio de Fisiología de La Conducta, Facultad de Medicina, Universidad de Los Andes, Mérida 5101, Venezuela
⁴ Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, USA

View larger version (53K): [in a new window]   Fig. 1. Expression and distribution of IP3 receptor isoforms in skeletal myotubes. (A) Representative western blots of six different experiments showing all three types of IP3Rs (IP3R1, IP3R2 and IP3R3) expressed in myotubes with a molecular mass of 260 kDa. No crossreactivity was seen. To correct for loading the blots were probed with an antibody that recognizes actin. (B) Upper panel, type-1 IP3R labeling is distributed in a dotted pattern in the nuclear envelope region. Inset, labelling of a differentiated myotube; striations correspond to limited regions of sarcoplasmic reticulum. Center panel, labeling of type-2 IP3R is distributed throughout the reticulum and absent from nuclei. Lower panel, labeling of type-3 IP3R is concentrated in nuclei, mainly in condensed structures and also occurs faintly in the cytoplasm. Bars, 5 µm (top), 25 µm (center, bottom).

View larger version (107K): [in a new window]   Fig. 2. Distribution of IP3 receptor isoforms in myonuclei. Co-immunofluorescence analysis of IP3 receptor isoforms with LAP 2 (a specific marker for the inner nuclear membrane). (A) Labels for both type-1 IP3R (upper image), and LAP 2 (center image) in the nuclear envelope were superimposed (yellow in bottom panel). (B) The labels for both type-2 IP3R (upper image) and LAP 2 (middle image) in the reticulum and nuclei respectively did not colocalize when images were superimposed (bottom image). (C) Immunolabel for type-3 IP3R (upper image) and LAP2 (middle image) show no colocalization (bottom image). Arrows show invaginations of the nuclear envelope towards the nucleoplasm colocalizing with type-1 IP3R, but not with either type-2 or type-3 IP3R. Bar, 10 µm.

View larger version (134K): [in a new window]   Fig. 3. Characterization of an isolated nuclear fraction from skeletal myotubes. (A) Fluorescence images of isolated myonuclei from a primary culture immunolabeled with LAP2 showing the integrity of the nuclei after the purification procedure. (B) Representative western blots of three different experiments showing minimal contamination of the nuclear fraction with the sarcoplasmic reticulum markers calsequestrin (top), and triadin (middle), but enriched with the nuclear marker LAP2 (bottom). (C) Transmission electron micrograph of a purified nucleus, showing the conserved nuclear structure. (D) High magnification electron micrograph showing the continuity and integrity of the nuclear membrane. Bar, 20 µm (A); 2 µm (C); 0.5 µm (D).

View larger version (54K): [in a new window]   Fig. 4. Expression of IP3 receptor isoforms in nuclear (N) and cytosolic (C) fractions of myotubes and their distribution in isolated nuclei. (A) Nuclear and cytosolic fractions of cultured myotubes bind [3H]IP3 with a Kd 82.55±20.29 nM and 91.2±11.4 nM, respectively, and have a maximal binding capacity of 2.41 and 1.64±0.10 pmol/mg protein [see Scatchard plots (insets)]. The non-specific binding was measured in the presence of 2 µM cold IP3. (B) Representative SDS-polyacrylamide gel analysis (from three independent preparations) of nuclear and cytosolic fractions. Type-1 IP3R was enriched in the nuclear fraction, but was also present in the cytosolic fraction (upper panel); type-2 IP3R was present only in the cytosolic fraction (middle panel), and type-3 IP3R was enriched in the nuclear fraction, but was also present in the cytosolic fraction (bottom panel). In all cases the same concentration of protein was loaded. (C,D) Immunofluorescence of isolated myonuclei revealed that type-1 IP3 receptor conserved the localization in the nuclear envelope (C) and that type-3 IP3R was distributed with a speckled pattern in the nucleoplasm (D). Bar, 15 µm.

View larger version (69K): [in a new window]   Fig. 5. Immunogold localization of IP3 receptors in isolated nuclei. Isolated nuclei from skeletal muscle cells were immunolabeled for type-1 or type-3 IP3R with isoform-specific antibodies linked to 10 nm gold particles. (A) Type-1 IP3R gold particles (indicated by arrows) located in the nuclear membrane mainly grouped in clusters. (B) Type-3 IP3R labeling (indicated by arrows) is located in groups of particles in the nuclear membrane and the nucleoplasm. Inset, the nucleolus presents clusters associated with undefined structures. Bar, 100 nm.

View larger version (33K): [in a new window]   Fig. 6. IP3 induced nucleoplasmic Ca2+ increases in isolated myonuclei. The time course of relative fluorescence change (ratio between the fluorescent difference, stimulated minus basal and the basal value) as a function of time is shown. (A) Stimulation of isolated myonuclei with 10 µM IP3 caused a quick and transient increase in calcium (). Such an increase was inhibited by 2-APB () and XeB (). The physiological solution (vehicle) alone did not induce any signal (). (B) Series of fluo-3 fluorescent images of myonuclei recorded at the times indicated. Basal fluorescence is shown in the top, the following image was taken immediately after 10 µM IP3 was added. (C) Isolated nuclei from 1B5 myotubes, stimulated with 10 µM IP3 display a quick and transient increase in calcium (), that was inhibited by 2-APB () and XeB (). The vehicle IP3 alone did not induce a signal (). (D) Series of fluo-3 fluorescent images in 1B5 nuclei collected at the times indicated. Basal fluorescence is shown at the top, the images that follow were taken after the stimulus with 10 µM IP3. (E) Fluorescence in a nucleus suspension using dyes reported to specifically locate to either the nuclear envelope (mag-fluo-4) or the nucleoplasm (fluo-4 dextran). The suspension was stimulated with 10 µM IP3 as indicated by the bar, traces shown are representative of 20 different measures from four different preparations. Nuclear diameter is approximately 10 µm.

View larger version (38K): [in a new window]   Fig. 7. IP3 and calcium induced CREB phosphorylation in isolated myonuclei. Top panel, western-blots of CREB phosphorylation at Ser133 following additions of Ca2+ and IP3 solutions at the times indicated. Incubation conditions were similar to those described for Fig. 6C. To correct for loading, blots were probed with an antibody that recognizes the phosphorylated and non-phosphorylated forms of CREB. Bottom panel, fold induction (mean±s.e.m. of three different experiments) of CREB phosphorylation over control levels. Significant differences *P<0.05 and **P<0.01 in induction of phosphorylation were observed between control and IP3- and Ca2+-treated groups at the indicated time points.