Fig. 4
Upon stress Rbfox1 aggregates in higher-order subcellular structures in ovaries. a, b Upon prolonged protein starvation (1–3 weeks) in Control (a) and miR-980 (b) ovaries, Rbfox1 aggregates in cytoplasmic and nuclear droplets of various size and shape. c, d Sometimes, Rbfox1 can form various phase-separated long fibers in both germline (c lower panel) and somatic follicle cells (d) of miR-980 mutants. e Similar higher order structures can be found in starved mitochondrial stress-linked parkin mutant ovaries. f, g Rbfox1-positive fibers (f) and round granules (g) formed in starved ovaries co-stain with Thioflavin T, a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils. h–j FRAP experiments performed on ovaries of flies with GFP-tagged Rbfox1 protein (Rbfox1CC00511/TM3). h Images of germline cell at different time points; prebleached (−1 s), postbleached (0 s), and after recovery (7 s). White box (ROI1) outlines an Rbfox1-positive granule subjected to photobleaching, yellow box (ROI2) outlines an Rbfox1-positive granule not subjected to photobleaching, and blue box (ROI3) outlines a background area that was not subjected to photobleaching (n > 50 measurments for each ROI). i Scatter plot for correlation analysis between the initial intensity, bleaching efficiency, mobile fraction, and recovery half-time parameters. j Fitted plots of the FRAP recovery curves show two distinct groups of Rbfox1-positive droplets that differ in their recovery half-times. Note that for FRAP experiments, due to lethality, heterozygous animals that have only one copy of GFP-tagged Rbfox1 protein were used (Rbfox1CC00511/TM3). This means that only half of the Rbfox1 proteins could be subjected to photobleaching and that non-labelled Rbfox1 proteins would also participate in liquid droplet recovery. Thus, the half-time of fluorescence recovery estimated here is most likely two times slower than the actual recovery half-time. DAPI (nuclei, magenta, a–e); Thioflavin T (amyloid, magenta, f, g); Rbfox1 (green, a–g). White rectangle in f, g outlines the area presented as separate panels (right), arrows point to Rbfox1-positive subcellular aggregates. a, b—ovarioles; c, f, g—egg chamber showing germline and somatic cells with large and small nuclei, respectively; d, e—follicular epithelial cells. Images in a–e are maximum intensity projections of multiple Z-sections, and images in f–h are single Z-sections. Scale bars 5 µm
