Fig. 3

BIN1iso1-induced degeneration is characterized by a strong accumulation of vesicles showing endosomal markers. a Electron microscopy images of retina expressing Luciferase (Ctrl), AmphA, BIN1iso1 and BIN1iso9 from 15-day-old flies. 1 to 2 ommatidia transversal sections are seen on the 4 left images. One photoreceptor neuron is outlined per image, its cytoplasm and nucleus being highlighted in yellow and its rhabdomere in green. Each ommatidium contains 6 outer photoreceptor neurons whose rhabdomeres are organized in a trapezoidal shape, with a 7th rhabdomere in the middle corresponding to one of the two inner photoreceptor neurons. In the BIN1iso1 condition, only 4 rhabdomeres can be seen. In addition, the cytoplasm of the photoreceptor is filled with vesicles, some of them (highlighted in magenta) are very big. These vesicles are surrounded by a single membrane (arrow on the right panel) as compared to the nuclear envelope (double arrow). Their rhabdomere is usually disintegrating (*). b Scheme of the organelles tested in photoreceptor neurons with the markers used. c, d Images of photoreceptor neurons expressing BIN1iso1 and EYFP-tagged endogenous Rab5 and GFP:2xFYVE, as early endosome markers (c), or EYFP-tagged endogenous Rab7 and evi:GFP, as late endosome/multivesicular body markers (d). Rh1 and actin staining, respectively yellow and magenta in the merge image, are used to label retina structure of 15-day-old flies. The photoreceptor neurons expressed Luciferase as a control or BIN1iso1. In the BIN1iso1 conditions, many vesicles were positive for the tested organelle markers (arrows, green staining in merge images). The extracellular inter-rhabdomeric staining for the evi:GFP marker (arrowhead) corresponds to exosomes. e Images of photoreceptor neurons expressing BIN1iso1 stained for BIN1, the photoreceptor neuron plasma membrane Na/K ATPase and actin in 7-day-old flies. BIN1iso1 is localized at the base of the rhabdomeres and around some abnormal vesicles as seen in the inset