Fig. 2.

Determining significance of association between MCP and endogenous MBS-containing β-actin mRNA. (A) Schematic representation of smFISH-IF on β-actin mRNP: 24 MBSs are present in β-actin 3′-UTR. Two MBSs separated by linker regions (gray) are illustrated for simplicity. Cy3-labeled RNA FISH probes (MBS probes are shown as red stars) hybridized to linker regions as described (18) are depicted. The MCP fused to GFP (gray circles and green barrels, respectively) is bound to the MBS as a dimer and can be detected by IF using antibodies against GFP and Alexa Fluor 647 (AF647)-conjugated secondary antibodies (illustrated with green stars). (B and C) Representative smFISH-IF images from dissociated hippocampal neurons from MBS mice expressing MCP-GFP by lentivirus infection were probed for (B) β-actin mRNA (MBS FISH probes, Cy3; red) or (C) CaMKII mRNA (CaMKII FISH probes, Cy3; red) and IF for MCP-GFP (GFP antibody, AF647; green). (B) A nonexpressing MCP-GFP neuron only showed FISH signal (red). MAP2 is shown in blue as a dendrite marker. (C) The image shows discrete fluorescent particles detected by both smFISH and IF throughout the dendrite that rarely overlap because the MCP does not bind CaMKII mRNA but binds β-actin mRNA with MBS in its 3′-UTR. Images are representative of four independent experiments, with over 15–20 dendrites observed in each experiment. (Scale bars: 5 μm.) (D) Schematic representation of a neuron and the super registration method that measures the significance of each mRNA–protein pair (red and green circles, respectively; magnified). The circle represents the nearest red circle (mRNA). The simulation measures the frequency that the number of green circles (protein) within this area would fall within distances less than d by chance. (Inset) The shaded area represents probability of chance association < 0.1 (the frequency for the illustrated pair based on 10,000 simulations). Every pair with this probability within 250 nm (the diffraction limit) is a single point in F and G. Complete data are in Fig. S2 E and F. (E) Curve of association between an mRNA and a binding protein was calculated as the cumulative ratio of association for intermolecular distances (in the range between 0 and 250 nm) that were less than a given observed distance. The ratio of association was calculated between the number of molecular pairs that can be found in proximity at each given nanometer of distance (and probability of chance association < 0.1) and the total number of molecular pairs within 250 nm (F and G). Red arrows show the distance wherein the mRNA–protein association for MCP-MBS and MCP-CaMKII is maximally separated [optimal distance (OD) = 69 nm] (Materials and Methods, Measurement of Association). Black line, MCP-MBS; dotted gray line, MCP-CaMKII. (F and G) Scatterplots show the probability of chance association between molecules for (F) MCP-GFP and β-actin mRNA (MBS) in MCP-MBS and (G) MCP-GFP and CaMKII mRNA (CaMKII) in MCP-CaMKII. Box A (pink) includes the associated molecules that have a probability of chance association < 0.1 and a distance less than the optimal distance of 69 nm (red vertical line; E). Box A includes the molecules that are physically likely to be in contact. Box B (light yellow) includes molecules with a probability of chance association < 0.1 but at distances greater than the optimal distance and within the diffraction limit of 250 nm. Box B includes the molecules that would be detected as positives by standard colocalization. The total numbers of intermolecular pairs in box A are 614 for MCP-MBS and 21 for MCP-CaMKII. The total numbers of pairs in box B are 120 for MCP-MBS and 111 for MCP-CaMKII (Fig. S2 E and F). (H) Distribution of observed distances for MCP-MBS (gray bars; Gaussian fit in red line) and MCP-CaMKII (MCP is bound to MBS on β-actin mRNA; black bars) after correction. Mean of observed distance was 34.58 ± 0.65 nm for MCP-MBS. Mean observed distance was 541.96 ± 8.14 nm for MCP-CaMKII (chance association) (Fig. S2D). Error, SEM.