Figure 3.
The interplay between the axon cortex and internal organelle determines the location of axon beads. DIV5–6 rat hippocampal neurons cultured in an AoC device were either transfected with Lifeact-GFP or cotransfected with both Lifeact-GFP and TagRFP-mito and live-imaged on DIV7–8 using a spinning disc inverted confocal microscope. (A) The localization of the mitochondria is compared with that of the beading areas. Asterisks indicate the beads containing mitochondria. Bar = 10 μm. (B) Line profile showing the fluorescence intensity of mitochondria (TagRFP-mito) and the axonal volume (Lifeact-GFP). Asterisks indicate the beads containing mitochondria in A. (C) Quantification of the percentage of beads containing mitochondria as shown in B (N = 21). (D) Representative time-lapse images capturing beads on axon branches, with locations marked by asterisks. Bar = 10 μm. (E) Quantification showing the percentage of beads formed on axon branches (N = 26). (F) Distribution of the spacing intervals between beads. (G) The cross-correlation between these intervals. The interval values were measured from 855 beads obtained from 42 axons. (H) Timeline depicting the measurement of mitochondria size (top) and speed (bottom). Size analysis was conducted at two key time points: at the start of the imaging session (0 s), ∼8 min before the initiation of flux, and at 952 s thereafter. Mobility measurements were conducted in three distinct spans of time: before (0–8 min), during (180 s), and after (from flux cessation to 10 min) the flux. (I) Time-lapse images show the instant shape changes of inner mitochondria induced by the low-speed flux (50 μl/min, 180 s). The boxed region is amplified in the right panels. Shape changes of the mitochondria are indicated with arrows. Bar = 10 μm (left), 10 μm (right top), and 5 μm (right bottom). (J) Paired comparison of mitochondria size fluctuations induced by low-speed flux (N = 9). (K) Axonal trafficking of the intra-axonal mitochondria during the 50 μl/min flux. The perpendicular lines in the kymograph indicate the pausing stage of the mitochondria, whereas the tilted slopes indicated by arrowheads are the mobile stage. x-axis bar = 10 μm; y-axis bar = 500 s. (L) Quantification of the average speed of the mobile mitochondrial trajectories of K (N = 328, 67, 275). (M) Paired comparison of mitochondrial speed for individual ROIs before, during, and after flux (N = 5 ROIs). Data represent mean ± SEM. Paired two-tailed Student’s t tests were applied in C, E, J, and M, and Welch’s t test in L; *P < 0.05, ***P < 0.001; n.s., non-significant.

The interplay between the axon cortex and internal organelle determines the location of axon beads. DIV5–6 rat hippocampal neurons cultured in an AoC device were either transfected with Lifeact-GFP or cotransfected with both Lifeact-GFP and TagRFP-mito and live-imaged on DIV7–8 using a spinning disc inverted confocal microscope. (A) The localization of the mitochondria is compared with that of the beading areas. Asterisks indicate the beads containing mitochondria. Bar = 10 μm. (B) Line profile showing the fluorescence intensity of mitochondria (TagRFP-mito) and the axonal volume (Lifeact-GFP). Asterisks indicate the beads containing mitochondria in A. (C) Quantification of the percentage of beads containing mitochondria as shown in B (N = 21). (D) Representative time-lapse images capturing beads on axon branches, with locations marked by asterisks. Bar = 10 μm. (E) Quantification showing the percentage of beads formed on axon branches (N = 26). (F) Distribution of the spacing intervals between beads. (G) The cross-correlation between these intervals. The interval values were measured from 855 beads obtained from 42 axons. (H) Timeline depicting the measurement of mitochondria size (top) and speed (bottom). Size analysis was conducted at two key time points: at the start of the imaging session (0 s), ∼8 min before the initiation of flux, and at 952 s thereafter. Mobility measurements were conducted in three distinct spans of time: before (0–8 min), during (180 s), and after (from flux cessation to 10 min) the flux. (I) Time-lapse images show the instant shape changes of inner mitochondria induced by the low-speed flux (50 μl/min, 180 s). The boxed region is amplified in the right panels. Shape changes of the mitochondria are indicated with arrows. Bar = 10 μm (left), 10 μm (right top), and 5 μm (right bottom). (J) Paired comparison of mitochondria size fluctuations induced by low-speed flux (N = 9). (K) Axonal trafficking of the intra-axonal mitochondria during the 50 μl/min flux. The perpendicular lines in the kymograph indicate the pausing stage of the mitochondria, whereas the tilted slopes indicated by arrowheads are the mobile stage. x-axis bar = 10 μm; y-axis bar = 500 s. (L) Quantification of the average speed of the mobile mitochondrial trajectories of K (N = 328, 67, 275). (M) Paired comparison of mitochondrial speed for individual ROIs before, during, and after flux (N = 5 ROIs). Data represent mean ± SEM. Paired two-tailed Student’s t tests were applied in C, E, J, and M, and Welch’s t test in L; *P < 0.05, ***P < 0.001; n.s., non-significant.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal