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Cytoplasmic trafficking of IGF-II mRNA-binding protein by conserved KH domains

¹ Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark
² Institute of Molecular Biology, University of Copenhagen, Denmark

View larger version (25K): [in a new window]   Fig. 1. RNA binding and subcellular distribution of GFP-IMP1. (A) UV crosslinking of IGF-II leader 3 segment C RNA with extract from either NIH 3T3 cells that stably express GFP-IMP1 (lane 1) or from normal NIH 3T3 cells (lane 2). (B) Cytoplasmic lysate from GFP-IMP1-expressing NIH 3T3 cells was separated in a 20-47% sucrose gradient. The upper panel shows the A260 sedimentation profile of ribosomal subunits and polyribosomes, and the lower panel shows the western analysis of the 12 fractions from the Mg2+-containing gradient with anti-IMP1 antibody to IMP1 and GFP-IMP1. M is a lane with recombinant IMP1.

View larger version (118K): [in a new window]   Fig. 2. Cytoplasmic localization of GFP-IMP1 during interphase and anaphase in NIH 3T3 cells. (A) Cytoplasmic localization of GFP-IMP1 during interphase. (1) A cell with a delocalised granular pattern of fluorescence that is typically seen in cells seeded on glass plates. (2) A migrating cell with GFP-IMP1 localized to the leading edge (arrow). (3,4) The GFP-IMP1-containing granules. (B) Time-lapse microscopy of GFP-IMP1 during mitosis displayed in glow scale.

View larger version (90K): [in a new window]   Fig. 3. GFP-IMP1 localization. (A) Time-lapse microscopy of NIH 3T3 cells that stably express GFP-IMP1. Cells with GFP-IMP1 undergoing localization are marked by arrows. (B) Velocity of moving GFP-IMP1 granules. Particles were followed by time-lapse microscopy at 20 second intervals. The velocities of 100 individual particles were calculated by measuring the distance between the particles in two succesive frames. (C) Fluorescence recovery after photobleaching. A cellular extension was bleached, and the transport of GFP-IMP1 into the bleached area was followed by time-lapse microscopy at 20 second intervals in the absence and presence of sodium azide and 2-deoxyglucose. A similar experiment with GFP-ß-galactosidase is included as a control.

View larger version (58K): [in a new window]   Fig. 4. GFP-IMP1 colocalises with microtubules and F-actin. NIH 3T3 cells that stably express GFP-IMP1 were fixed and stained with anti-tubulin antibody or anti-vimentin and phalloidin and examined by confocal microscopy. (A) The simultaneous detection of GFP-IMP1 (green) and microtubules (red) and their colocalization (arrow). (B, upper panel) The simultaneous detection of GFP-IMP1 (green), microfilaments (red) and microtubules (blue). The arrow in the overlay frame indicates the colocalization of F-actin and GFP-IMP in the lamellipodia. (B, lower panel) The detection of GFP-IMP1 (green), vimentin (blue) and microfilaments (red). (C, upper panel) Cells treated with nocodazole. Most GFP-IMP1 collapses into large aggregates, but a fraction of GFP-IMP1 remains anchored in the lamellipodia for more than 1 hour. (C, lower panel) Time-lapse microscopy of GFP-IMP1 after cytochalasin D treatment. At these short time points, the integrity of the cell is intact, but anchoring in lamellipodia has disappeared.

View larger version (60K): [in a new window]   Fig. 5. IMP1 binds to microtubules. (A) 32P-labelled recombinant IMP1-HMK was precipitated with tubulin and microtubule-associated proteins (MAPs) in the presence of paclitaxel and GTP. IMP1-HMK in the presence of buffer (lane 1); polymerised tubulin (lane 2); polymerised tubulin and 1.25 µg total MAPs (lane 3); polymerised tubulin and 2.5 µg total MAPs (lane 4); polymerised tubulin and 5 µg total MAPs and 15 nM leader 3 segment C RNA (lane 5); unpolymerised tubulin and 2.5 µg total MAPs (lane 6); polymerised tubulin and 2.5 µg total MAPs and 2 units RNase T1 (lane 7); polymerised tubulin and 3 µg MAP2 (lane 8). The autoradiograph is on the left, and the Coomassie brilliant blue staining is on the right. M is a molecular mass marker. (B) Pull-down of 32P-labelled IMP1-HMK in the absence of polymerised actin (lane 1, supernatant; lane 2, pellet) or in the presence of polymerised actin (lane 3, supernatant; lane 4, pellet). Pull-down of actinin by polymerised actin (lane 5, supernatant; lane 6, pellet). The autoradiograph is on the left, and the Coomassie brilliant blue-staining is on the right.

View larger version (52K): [in a new window]   Fig. 6. CFP-IMP1 does not colocalise with membranous compartments. (A) CFP-IMP1 was cotransfected into NIH 3T3 cells together with YFP-linked markers of the endoplasmic reticulum (YFP-KDEL) and Golgi apparatus (YFP-ß1,4GT), or GFP-IMP1 was cotransfected with a Texas Red-linked marker for acid vesicles. There is no apparent colocalization of fluorescently tagged IMP1 with any of the markers and, in the cases of the Golgi apparatus and acid vesicles, exclusion is often observed. The pictures from cotransfections with ER and Golgi markers are in pseudo-colours to aid visualisation: YFP fluorescence is shown in red, CFP fluorescence is shown in green, and regions of overlap are shown in yellow. (B, upper and middle panels) Western blot with anti-calnexin and anti-IMP1 antibodies of 12 fractions from a 10-60% Nycodenz sedimentation analysis of an undiluted RD cellular lysate. Positions of molecular mass markers are shown at the left. (B, lower panel) Slot-blot analysis of total RNA from the same fractions with a probe specific for IGF-II leader 3 mRNA.

View larger version (50K): [in a new window]   Fig. 7. Subcytoplasmic localization of IMP1 is mediated by the four KH domains. NIH 3T3 cells were transiently transfected with GFP-IMP1 deletion constructs. (A) Schematic representation of the constructs and their ability (+) to form granules and localise subcytoplasmically. *The KH2-4 construct exhibited a tendency to form immobile aggregates that could be mistaken for granules. (B) Representative pictures of the constructs. (C) Cotransfection of CFP-IMP1 and YFP-KH1-4. The frames are in pseudo-colours to aid visualisation: CFP fluorescence is shown in green, YFP fluorescence is shown in red, and regions of overlap are shown in yellow.

View larger version (48K): [in a new window]   Fig. 8. RNA binding and MAP association are mediated by the KH modules. Autoradiograph from an electrophoretic mobility-shift analysis of 32P-labelled H19 segment H (H19 seg. H) with recombinant IMP-1 (lane 2: 0.3 nM; lane 3: 3 nM; lane 9: 0.6 nM), RRM1-2 (lane 4: 10 nM; lane 5: 100 nM), KH1-4 (lane 6: 0.6 nM; lane 7: 1.2 nM; lane 8: 3 nM), KH1-2 (lane 10: 20 nM; lane 11: 100 nM) and KH3-4 (lane 12: 20 nM; lane 13: 100 nM). The positions of unbound RNA and RNA-protein complexes a and b are shown at the left. (B) Real-time surface plasmon resonance binding of phosphocellulose-purified MAPs to recombinant RRM1-2 and KH1-4 immobilized on a CM5 sensorchip surface (left panel), and binding of phosphocellulose-purified MAPs and MAP-enriched tubulin to immobilized Drosophila IMP (right panel).