Figure 1. Loss of JIP3 results in the... | Rockefeller University Press

Figure 1.

$Figure 1. Loss of JIP3 results in the axonal accumulation of lysosomes. (A and B) LAMP1 immunofluorescence in WT and JIP3 KO cortical neuron primary cultures (12 DIV). Arrows indicate position of cell bodies. Bar, 10 µm. (C and D) Live-cell images of LysoTracker fluorescence in WT and JIP3 KO axons of primary neurons grown at low density. Arrowheads highlight individual lysosomes in the WT axon and focal axonal swellings filled with lysosomes in the JIP3 KO. Bar, 10 µm. (E) Quantification of the percentage of neurons with lysosome-filled axonal swellings (10 DIV, low-density cultures, mean ± SD from three independent KO and control cultures, >30 neurons per genotype; ***, P < 0.001, unpaired t test). (F) Axonal LysoTracker signals in WT and JIP3 KO (outside of swellings) neurons. Bar, 5 µm. (G) Quantification of lysosome density in WT and JIP3 KO axons (outside of swellings; 15–17 neurons per genotype from 3 independent cultures; mean ± SD; *, P = 0.0118, unpaired t test). (H) Quantification of fraction of stationary axonal lysosomes in WT and JIP3 KO (outside of swellings) neurons (mean ± SD); n = 3 independent WT and JIP3 KO littermate cultures; ∼90 WT lysosomes and 150 JIP3 KO lysosomes analyzed; **, P < 0.01, unpaired t test). (I) LAMP1 and neurofilament immunofluorescence confirm the axonal localization of lysosome accumulations in the JIP3 KO neurons. Bar, 10 µm. (J and K) Stitched images of VAMP2 (green; presynaptic protein), LAMP1 (red; lysosomes), and MAP2 (blue; dendrites) immunofluorescence in WT and JIP3 KO neurons reveals that sites of lysosome accumulation are positive for the presynaptic marker. Bars: 25 µm; (inset) 1 µm.$

Loss of JIP3 results in the axonal accumulation of lysosomes. (A and B) LAMP1 immunofluorescence in WT and JIP3 KO cortical neuron primary cultures (12 DIV). Arrows indicate position of cell bodies. Bar, 10 µm. (C and D) Live-cell images of LysoTracker fluorescence in WT and JIP3 KO axons of primary neurons grown at low density. Arrowheads highlight individual lysosomes in the WT axon and focal axonal swellings filled with lysosomes in the JIP3 KO. Bar, 10 µm. (E) Quantification of the percentage of neurons with lysosome-filled axonal swellings (10 DIV, low-density cultures, mean ± SD from three independent KO and control cultures, >30 neurons per genotype; ^***, P < 0.001, unpaired t test). (F) Axonal LysoTracker signals in WT and JIP3 KO (outside of swellings) neurons. Bar, 5 µm. (G) Quantification of lysosome density in WT and JIP3 KO axons (outside of swellings; 15–17 neurons per genotype from 3 independent cultures; mean ± SD; *, P = 0.0118, unpaired t test). (H) Quantification of fraction of stationary axonal lysosomes in WT and JIP3 KO (outside of swellings) neurons (mean ± SD); n = 3 independent WT and JIP3 KO littermate cultures; ∼90 WT lysosomes and 150 JIP3 KO lysosomes analyzed; ^**, P < 0.01, unpaired t test). (I) LAMP1 and neurofilament immunofluorescence confirm the axonal localization of lysosome accumulations in the JIP3 KO neurons. Bar, 10 µm. (J and K) Stitched images of VAMP2 (green; presynaptic protein), LAMP1 (red; lysosomes), and MAP2 (blue; dendrites) immunofluorescence in WT and JIP3 KO neurons reveals that sites of lysosome accumulation are positive for the presynaptic marker. Bars: 25 µm; (inset) 1 µm.