Axonal precursor miRNAs hitchhike on endosomes and locally regulate the development of neural circuits (2020)
Figure 4

Figure 4.

pre-miR-181a-1 is trafficked docked to late endosomes / lysosomes

A. Schematic representation of the experimental paradigm. Plasmid concentrations used: 0.5μg/μL pCS2-CD63-eGFP, pCS2-CD63-mRFP, pCS2-Rab7a-mRFP or pCS2-Lamp1-eGFP; 0.4μg/μL pCS2-Rab7a-eGFP or pCS2-Rab5a-mRFP; 0.3 μg/μL pCS2-Rab5a-eGFP; 50nM LysoTracker; 200ng/μL cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1.

B. Overview of the markers used. Figure adapted from (Yap et al, 2018). Abbreviations: EE, early endosome; LE, late endosome; Ly, lysosome.

C. Representative images of co-electroporated axons. Dashed white lines delineate axons. White arrows indicate colocalized puncta. Colored arrows represent non-colocalized signal as follows: magenta for CD63 and yellow for Lamp1 or Rab5a. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP. Scale bars: 5μm.

D. Frequency (in percentage) of CD63 colocalization with specific organelle markers. Each data point corresponds to one axon. Total number of puncta, axons, and independent experiments analyzed: 540 puncta, 23 axons, n=4 (CD63/Rab7a), 416 puncta, 18 axons, n=3 (CD63/LysoTracker), 584 puncta, 17 axons, n=2 (CD63/Lamp1), 649 puncta, 19 axons, n=3 (CD63/Rab5a). Values are mean ± SEM. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP.

E. Representative images of co-electroporated axons. Dashed white lines delineate axons. White arrows indicate colocalized puncta. Colored arrows represent non-colocalized signal as follows: cyan for pre-miR-181a-1 and yellow for Lamp1 or Rab5a. Abbreviations: Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1. Scale bars: 5μm.

F. Frequency (in percentage) of pre-miR-181a-1 colocalization with specific organelle markers. Each data point corresponds to one axon. Total number of puncta, axons and independent experiments analyzed: 825 puncta, 41 axons, n=8 (pre-miR-181a-1/CD63), 732 puncta, 33 axons, n=7 (pre-miR-181a-1/Rab7a), 681 puncta, 29 axons, n=5 (pre-miR-181a-1/LysoTracker), 501 puncta, 17 axons, n=2 (pre-miR-181a-1/Lamp1), 791 puncta, 25 axons, n=3 (pre-miR-181a-1/Rab5a). Values are median with interquartile range. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP, Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1.

G. Representative images of co-electroporated axons. Dashed white lines delineate axons. White arrows indicate colocalized puncta. Colored arrows represent non-colocalized signal as follows: magenta for CD63, cyan for pre-miR-181a-1, yellow for Rab7a and blue for LysoTracker. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1. Scale bar: 5μm.

H. Frequency (in percentage) of pre-miR-181a-1 colocalization with vesicles double positive for organelle markers. Each data point corresponds to one axon. Total number of puncta, axons and independent experiments analyzed: 745 puncta, 23 axons, n=5 (CD63/pre-miR-181a-1/Rab7a), 577 puncta, 18 axons, n=3 (CD63/pre-miR-181a-1/LysoTracker). Values are median with interquartile range. Abbreviations: ns, not significant; CD63, CD63-eGFP or CD63-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1.

I. Representative 3D-STED super-resolution image. Total number of puncta, axons and growth cones analyzed: 99 puncta, 16 axons and 10 growth cones (MB), 291 puncta, 36 axons and 15 growth cones (cy3-pre-miR-181a-1). n=2 (MB), n=3 (cy3-pre-miR-181a-1) independent experiments. Abbreviations: CD63, CD63-eGFP; pre-miR-181a-1, cy3-pre-miR-181a-1; MB; molecular beacon. Scale bars: 200nm.

Data information: **** p<0.0001. Values were normally distributed (Shapiro-Wilk test). One-way ANOVA with Tukey's multiple comparison post-hoc test (D). Values were not normally distributed (Shapiro-Wilk test) (F,H). Kruskal-Wallis test with Dunn's multiple comparison post-hoc test (F). Two-tailed Mann Whitney test (H).