Fig 2: EndophilinA2 and A3 are recruited to endosomes in response to BDNF. (A) Representative TIRF microscopy images of MEFs co-transfected with GFP-tagged EndophilinA2 (left panels) or EndophilinA3 (right panels) and the RFP-tagged endosomal markers EEA1, Rab5, Rab7 or Lamp1, in the presence and absence of 100 ng/ml BDNF. Arrows indicate colocalization of EndophilinA (green) with endosomal markers (red). (B) Quantitation of colocalization of transfected GFP-tagged EndophilinA2, and A3 with RFP-tagged EEA1, Rab5, Rab7 and Lamp1; n = 8–10 images per condition from 3 independent cell cultures, significance determined by unpaired two-tailed Student’s t-test test comparing non-stimulated to stimulated conditions, error = SEM, *p < 0.05, **p < 0.01, ***p < 0.001, scale bar = 10 µm.

Fig 3: Rab7-positive endosomes accumulate in EndophilinA TKO hippocampal neurons. (A) Identification of endosomal structures in wild-type and EndophilinA TKO neurons using immunocytochemistry to detect endogenous levels of the endosomal markers EEA1, and Rab7. Representative images of WT and Endophilin TKO hippocampal neurons stained for EEA1 and Rab7 are shown. Map2 antibody was used to label neurons. Scale bar = 10 μm; (B) Quantification of endosomal marker signal for EEA1 or Rab7 (C) normalized to area. Significance determined by unpaired two-tailed Student’s t-test comparing each condition to control, n = 30 images per condition from 3 independent cell cultures; error = SEM, **p < 0.01. (C) Western blots showing levels of EEA1 and Rab7 in wild-type and EndophilinA TKO brain homogenates. (D) Quantitation of protein levels of EEA1 and Rab7 (F) in control and EndophilinA TKOs normalized to actin; n = 3 independent brain samples, error = SEM, *p < 0.05, **p < 0.01.

Fig 4: EndophilinAs tubulate endosomal compartments to which they are recruited. (A) Representative timecourse of a tubulation event of EndophilinA2 on a Rab7-positive endosome over 200 seconds in co-transfected EndophilinA TKO MEFs. Scale bar = 5 µm. (B) Example of tubulation events of EndophilinA1 on Lamp1-positive endosomes in the absence (NF) and presence of 100 ng/ml BDNF, of EndophilinA2 on Rab7 and Lamp1-positive endosomes in the absence (NF) and presence of BDNF (C), and of EndophilinA3 on EEA1, Rab7, and Lamp1-positive endosomes in the absence (NF) and presence of BDNF (D) Arrows indicate tubulation events in which endosomal markers and EndophilinA isoforms co-localize. Scale bars = 2 µm for (B–D). (E) Quantitation of the percent increase in tubulation of endosomal compartments by EndophilinA1, EndophilinA2, and EndophilinA3, compared to EndophilinA triple knockouts in control no factor (NF) conditions and following 100 ng/ml BDNF treatment; n = 8–10 images per condition from 3 independent cell cultures, significance determined by paired two-tailed Student’s t-test, error = SEM, *p < 0.05, **p < 0.01, ***p < 0.001, scale bar = 10 µm.

Fig 5: EndophilinA2 and EndophilinA3 co-traffic with endosomes. (A) Kymographs (left panels) of co-trafficking of EndophilinA2 and endosomal markers before and after application of 100 ng/ml BDNF in 5 minute TIRF time-lapse images of MEFs co-transfected with GFP-tagged EndophilinA2 and RFP-tagged EEA1, Rab5 (B), Rab7 (C), or Lamp1 (D). Quantitation of recruitment of EndophilinA2 to endosomes and mobility of EndophilinA2-positive endosomes (before or after administering 100 ng/ml BDNF) by percent of EndophilinA2 and endosomal kymograph tracks colocalizing, and percent of co-localizing mobile endosomes (A–D, right panels). (E) Kymographs of co-trafficking of EndophilinA3 and RFP-tagged Rab5 (E), Rab7 (F), or Lamp1 (G). Quantitation of recruitment of EndophilinA3 to endosomes before and after administering BDNF, by percent of tracks colocalizing, and percent of co-localizing mobile endosomes (E–G, right panels); n = 8–10 images per condition from 3 independent cell cultures, significance determined by unpaired two-tailed Student’s t-test non-stimulated to stimulated conditions, error = SEM, *p < 0.05, **p < 0.01, ***p < 0.001, scale bar = 10 µm.