Fig. 5

Assembly of mRNP into puncta in different cellular states correlates with levels of protein synthesis. a Representative immunofluorescence images showing Fmrp (green) and Dcp1a (red) puncta in G0, MB, and MT, as well as cells reactivated for 3 h from G0 (R3). Arrows indicate prominent puncta. Notably, Fmrp puncta are large and prominent in G0, disperse at 3 h post reactivation and are less evident in asynchronous MB. Dcp1a puncta are nearly absent in G0 and reappear at R3; Dcp1 puncta are also more prominent in MB than MT. Fmrp and Dcp1a mostly localize to distinct puncta; the rare yellow puncta seen in R3 and MT may reflect transient co-localization due to passage of transcripts between two types of mRNP granules as inferred in [45]. b Measurement of the rate of protein synthesis using OPP incorporation into newly synthesized proteins reveals active translation in MB and MT, and substantial suppression in G0. c Quantification of images in b. Fluorescent intensity was measured in 150 cells from each condition, the box and whisker plot shows integrated fluorescence for each cell (each dot represents one cell), limits on the box correspond to 75th and 25th percentile values. “Mb-neg” and “G0-neg” represent samples of MB and G0 that were not pulsed with OPP but processed for detection along with samples that were exposed to OPP. N = 2 biological replicates. Data were analyzed by multivariate ANOVA with post hoc HSD Tukey tests performed for each pairwise comparison. *** p < 0.0001, ** p < 0.001. d Immunolabeling of translation initiation factors eIF-4E (red) and eIF-4G (pink) in G0, R3, MB, and MT: Upper panel shows merged images, and lower panels show detection of each factor individually. Expression and assembly of these translation factors correlates with levels of protein synthesis seen in b and c: poor in G0, restored assembly with distinct puncta in R3, and strong expression and organization of eIF-4E and eIF-4G complexes in MB and MT