Figure 4. GFP-JIP3 localizes to axonal... | Rockefeller University Press

Figure 4.

$Figure 4. GFP-JIP3 localizes to axonal lysosomes and rescues axonal lysosomes over abundance in JIP3 KO neurons. (A and B) Representative stitched image of 9 DIV JIP3 KO neurons transfected at 7 DIV with GFP and Cherry-Rab7 (A) or GFP-JIP3 and Cherry-Rab7 (B) showing that GFP-transfected KO neurons have large axonal swellings filled with lysosomes (A; yellow box with dashed lines), whereas the majority of GFP-JIP3–transfected KO neurons do not exhibit this phenotype. Bars: 25 µm; (inset) 5 µm. (C) Quantification of fraction of neurons showing one or more lysosome-filled axonal swellings in GFP or GFP-JIP3–transfected JIP3 KO neurons (n = 5 independent experiments, mean ± SD; 35–45 neurons per treatment/transfection type; ***, P < 0.001). (D and E) Portions of WT axons transfected with either GFP-JIP3 and Cherry-LAMP1 (D) or GFP-JIP3 and Cherry-Rab7 (E) showing enrichment of GFP-JIP3 (green arrowheads) on lysosomes (red arrowheads). Bars, 5 µm. (F) Image of GFP-JIP3–transfected WT axon showing GFP-JIP3 distributed in vesicles and local accumulation at axon tips and branches (arrowheads highlight JIP3-positive vesicles). Bar, 10 µm. (G) High levels of GFP-JIP3 overexpression induce the formation of abnormal axonal swellings (asterisk) even in WT neurons. Bar, 10 µm.$

GFP-JIP3 localizes to axonal lysosomes and rescues axonal lysosomes over abundance in JIP3 KO neurons. (A and B) Representative stitched image of 9 DIV JIP3 KO neurons transfected at 7 DIV with GFP and Cherry-Rab7 (A) or GFP-JIP3 and Cherry-Rab7 (B) showing that GFP-transfected KO neurons have large axonal swellings filled with lysosomes (A; yellow box with dashed lines), whereas the majority of GFP-JIP3–transfected KO neurons do not exhibit this phenotype. Bars: 25 µm; (inset) 5 µm. (C) Quantification of fraction of neurons showing one or more lysosome-filled axonal swellings in GFP or GFP-JIP3–transfected JIP3 KO neurons (n = 5 independent experiments, mean ± SD; 35–45 neurons per treatment/transfection type; ***, P < 0.001). (D and E) Portions of WT axons transfected with either GFP-JIP3 and Cherry-LAMP1 (D) or GFP-JIP3 and Cherry-Rab7 (E) showing enrichment of GFP-JIP3 (green arrowheads) on lysosomes (red arrowheads). Bars, 5 µm. (F) Image of GFP-JIP3–transfected WT axon showing GFP-JIP3 distributed in vesicles and local accumulation at axon tips and branches (arrowheads highlight JIP3-positive vesicles). Bar, 10 µm. (G) High levels of GFP-JIP3 overexpression induce the formation of abnormal axonal swellings (asterisk) even in WT neurons. Bar, 10 µm.