Figure 3.
The NRG3 NTF is endocytosed from the PM. (A) Airyscan high-resolution time-lapse images of GFP-proNRG3 transfected HEK293 cells showing endocytosis of the NTF over the course of 30 s (red arrowhead; see also Video 1). (B) Schematic illustration of the experimental approach used to monitor NRG3 NTF endocytosis. Top: GFP-tagged proNRG3 and BACE1 processed NRG3 NTF harboring a BBS upstream of the EGF-L domain (denoted GFP-proNRG3BBS and GFP-NRG3BBS NTF, respectively). Bottom: Schematic illustration showing surface uptake and internalization of fluorescently labeled BTX (BTX-A555) by the GFP-NRG3BBS NTF. Note that in experiments including GFP-tagged SNPH (see H and I), a proNRG3BBS variant without GFP but with a V5 tag upstream of the EGF-L domain was used instead. However, for consistency across panels, NRG3 is always shown in green, with other markers shown in red (BTX, Rab5) or magenta (SNPH). (C) Western blot of transfected HEK293 cells showing BACE1 dependent GFP-proNRG3BBS processing. Note how BACE1 inhibition by BACE-IV reduces NTF signals and causes the accumulation of the unprocessed proform detected with antibodies against GFP (top) and the NRG3 ICDC (bottom). (D) Still frames from live-cell imaging of HEK293 cells expressing GFP-proNRG3 (top) or GFP-proNRG3BBS (bottom). Cells were surface-labeled with BTX–Alexa 555 for 30 min prior to imaging. Note the extensive overlap between BTX and GFP signals for GFP-proNRG3BBS, but not for the negative control GFP-proNRG3, in the micrographs and the corresponding densitometric line scans. The summary graph shows GFP and BTX–Alexa 555 colocalization (right). Data are plotted as Mander’s overlap coefficient and represent the mean ± SEM from three independent experiments (n = 8 cells). (E) Representative image and line scan densitometry illustrating extensive NRG3/Rab5 colocalization in a HEK293 cell co-transfected with V5-tagged proNRG3 and GFP-tagged Rab5 (arrowheads). Single-channel images shown in grayscale. (F) Representative image of a neuron transfected with GFP-proNRG3BBS and labeled for 30 min with BTX–Alexa 555. The magnified area in the lower panel shows a primary neurite, with single-channel images shown in grayscale. Note the extensive overlap between GFP and BTX–Alexa 555 indicative of recent NRG3 NTF endocytosis. Similar results were obtained in three independent experiments. (G) Representative overview image and magnified area of a GFP-proNRG3BBS–transfected and BTX–Alexa 555-treated neuron (5 min), additionally labeled with anti-MAP2 to demonstrate NRG3 endocytosis in dendrites. Location of the densitometric line scan shown on the right is indicated in the merged magnified image. (H) Similarly, neurons were transfected with V5-tagged NRG3BBS and GFP-SNPH, incubated for 5 or 30 min with BTX–Alexa 555, fixed, and labeled with anti-V5. Note that very little BTX signal was detected in SNPH+ axons after 5 min whereas double NTF+/BTX+ puncta were abundant after 30 min. (I) Quantitative co-localization analysis of experiments shown in G and H confirms extensive overlap of BTX and NRG3 NTF signals in MAP2+ dendrites at 5 min, and in SNPH+ axons at 30 min but not 5 min, indicating that transcytosis of the NRG3 NTF occurs chiefly in the somatodendritic compartment. Data plotted as Mander’s overlap coefficients, representing the mean ± SEM from three independent experiments (n = 16 ROIs). ****, P < 0.0001 (unpaired t test). Scale bars: A, E, H, and F and G (insets), 5 μm; D, 10 μm; F and G, 20 µm. Source data are available for this figure: SourceData F3.

The NRG3 NTF is endocytosed from the PM. (A) Airyscan high-resolution time-lapse images of GFP-proNRG3 transfected HEK293 cells showing endocytosis of the NTF over the course of 30 s (red arrowhead; see also Video 1). (B) Schematic illustration of the experimental approach used to monitor NRG3 NTF endocytosis. Top: GFP-tagged proNRG3 and BACE1 processed NRG3 NTF harboring a BBS upstream of the EGF-L domain (denoted GFP-proNRG3BBS and GFP-NRG3BBS NTF, respectively). Bottom: Schematic illustration showing surface uptake and internalization of fluorescently labeled BTX (BTX-A555) by the GFP-NRG3BBS NTF. Note that in experiments including GFP-tagged SNPH (see H and I), a proNRG3BBS variant without GFP but with a V5 tag upstream of the EGF-L domain was used instead. However, for consistency across panels, NRG3 is always shown in green, with other markers shown in red (BTX, Rab5) or magenta (SNPH). (C) Western blot of transfected HEK293 cells showing BACE1 dependent GFP-proNRG3BBS processing. Note how BACE1 inhibition by BACE-IV reduces NTF signals and causes the accumulation of the unprocessed proform detected with antibodies against GFP (top) and the NRG3 ICDC (bottom). (D) Still frames from live-cell imaging of HEK293 cells expressing GFP-proNRG3 (top) or GFP-proNRG3BBS (bottom). Cells were surface-labeled with BTX–Alexa 555 for 30 min prior to imaging. Note the extensive overlap between BTX and GFP signals for GFP-proNRG3BBS, but not for the negative control GFP-proNRG3, in the micrographs and the corresponding densitometric line scans. The summary graph shows GFP and BTX–Alexa 555 colocalization (right). Data are plotted as Mander’s overlap coefficient and represent the mean ± SEM from three independent experiments (n = 8 cells). (E) Representative image and line scan densitometry illustrating extensive NRG3/Rab5 colocalization in a HEK293 cell co-transfected with V5-tagged proNRG3 and GFP-tagged Rab5 (arrowheads). Single-channel images shown in grayscale. (F) Representative image of a neuron transfected with GFP-proNRG3BBS and labeled for 30 min with BTX–Alexa 555. The magnified area in the lower panel shows a primary neurite, with single-channel images shown in grayscale. Note the extensive overlap between GFP and BTX–Alexa 555 indicative of recent NRG3 NTF endocytosis. Similar results were obtained in three independent experiments. (G) Representative overview image and magnified area of a GFP-proNRG3BBS–transfected and BTX–Alexa 555-treated neuron (5 min), additionally labeled with anti-MAP2 to demonstrate NRG3 endocytosis in dendrites. Location of the densitometric line scan shown on the right is indicated in the merged magnified image. (H) Similarly, neurons were transfected with V5-tagged NRG3BBS and GFP-SNPH, incubated for 5 or 30 min with BTX–Alexa 555, fixed, and labeled with anti-V5. Note that very little BTX signal was detected in SNPH+ axons after 5 min whereas double NTF+/BTX+ puncta were abundant after 30 min. (I) Quantitative co-localization analysis of experiments shown in G and H confirms extensive overlap of BTX and NRG3 NTF signals in MAP2+ dendrites at 5 min, and in SNPH+ axons at 30 min but not 5 min, indicating that transcytosis of the NRG3 NTF occurs chiefly in the somatodendritic compartment. Data plotted as Mander’s overlap coefficients, representing the mean ± SEM from three independent experiments (n = 16 ROIs). ****, P < 0.0001 (unpaired t test). Scale bars: A, E, H, and F and G (insets), 5 μm; D, 10 μm; F and G, 20 µm. Source data are available for this figure: SourceData F3.

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