Fig. 4: RNAs accumulate in the perinuclear region of myofibers after microtubule ablation.
From: Microtubule-based transport is essential to distribute RNA and nascent protein in skeletal muscle
A Schematic describing microtubule depolymerization experiment. B IF of tubulin (green) and telethonin (red) protein in myofibers from A. Scale bars: 5 µm. C Representative FISH images of Ttn RNA in myofibers from A. Scale bars: 5 µm. D Same as C but Polr2a RNA. Scale bars: 5 µm. E Intranuclear position relative to centroid (C) and periphery (P) for intranuclear spots and distance to nearest nucleus for cytoplasmic spots; shown for Ttn RNA. Bar colors correspond to treatment condition and shaded area denotes perinuclear region, defined as <2 μm from nuclear periphery (n.p.). Number of myofibers per RNA and condition are Dmd: DMSO-8, nocodazole-8, washout-6; Gapdh: DMSO-9, nocodazole-8, washout-3; Hist1h1c: DMSO-7, nocodazole-12, washout-4; Hnrnpa2b1: DMSO-8, nocodazole-5, washout-5; Myom1: DMSO-5, nocodazole-5, washout-5; Polr2a: DMSO-5, nocodazole-5, washout-5; Ttn: DMSO-11, nocodazole-8, washout-5; Vcl: DMSO-5, nocodazole-5, washout-5. F Same as E but Polr2a RNA. G Fraction of total spots in the perinuclear region for each gene. Colors as in E. Bars are mean ± SEM. Black dots are individual images. Number of myofibers as in E. *p < 0.05, Two-sided Mann–Whitney U test. H Representative images for each RNA showing zoomed-in regions of individual nuclei. Scale bars: 1 µm. I Cytoplasmic and perinuclear blob intensities for Gapdh RNA in each experimental condition. Colors as in E. J Intensity of perinuclear blobs. Bars are 95th percentile ±95% CI. n.s. not significant, *p < 0.05, by confidence interval overlap. Confidence intervals were estimated by bootstrapping. K Same as J but for cytoplasmic blobs. Number of myofibers as in E. L Schematic describing the effects of Actinomycin D and nocodazole treatments on cytoplasmic RNA concentration. M For each RNA, the fraction remaining in the cytoplasm after 18 h nocodazole treatment compared with the fraction predicted by our decay model. Trend line: LLS regression, slope = 0.84; pearson R = 0.66; p < 0.05, Wald test.
