Syntaphilin-Mediated Docking of Mitochondria at the Growth Cone Is Dispensable for Axon Elongation In Vivo

Mitochondrial distribution changes with growth cone behavior. A, Representative time-lapse images of mitochondria (mitoEGFP, white) in distal retinal axons and growth cones (labeled with TagRFP, delineated in orange and shown in left panels) classified as pausing or advancing (see Movies 2, 3, 4). Most growth cones alternate periods of pausing and advancing/elongating, resulting in net axon growth, as shown in the bottom panels. The red asterisk indicates mitochondria in neighboring axons. Scale bar, 5 μm. B–C´, Quantification of the mitochondrial occupancy (as a percentage) of the growth cone total volume (B), total area (B´), central volume (C), and central area (C´). D, D´, Quantification of the mitochondrial volume in the peripheral growth cone (D) and of the number of mitochondria in the growth cone peripheral area (D´). E, Quantification of the distance between the largest mitochondrial cluster and the growth cone leading edge. When growth cones elongate, the distance to the growth cone leading edge increases significantly. Data from 12 independent experiments (pausing, n = 11; advancing, n = 8) are shown as mean ± SEM. Statistical analysis (B–E): unpaired t test, ***p < 0.001.

Pioneering mitochondrial clusters localize near the leading edge in advancing growth cones. A, Representative time-lapse images of mitochondria (mitoEGFP, white) in an advancing growth cone (Movie 5). While the main mitochondrial cluster (green arrow) lags behind during growth cone advance, some small pioneering mitochondrial clusters (blue arrows) appear in close proximity to the leading edge. Lateral view, confocal maximal projections. Scale bar, 3 μm. B, C, EGFP intensity profiles calculated along a line between the initial proximal growth cone and final leading edge to analyze the distribution of mitochondria along the advancing growth cone at successive time points shown in A. C, The orange and green lines correspond to the fluorescent intensities of mitochondria in the growth cone and at the proximal growth cone, respectively (see B: Analysis).

Anterograde mitochondrial transport correlates with axonal outgrowth. A, Representative kymograph of mitochondria (mitoEGFP, white) in a distal axon whose growth cone alternates between advancing and pausing. The first and last frames of the time-lapse recording (Movie 4) are shown with axon and growth cone delineated in orange. Confocal maximal projections. Scale bar, 5 μm. The growth cone leading edge and proximal growth cone (green line) are indicated on the kymograph and kymograph analysis panels. B, Quantification of net transport, analyzed by counting the number of mitochondria moving anterogradely or retrogradely. Data from 12 independent experiments (pausing: n = 11, advancing: n = 8) are shown as the mean ± SEM. Statistical analysis: two-way ANOVA with post hoc Bonferroni test, **p < 0.01, ***p < 0.001. C, Quantification of the percentage of time mitochondria spent in a mobile or stationary state. A significant increase in time spent in motion (grey asterisk, p = 0.013) and a decrease in time spent in a stationary state (red asterisk, p = 0.008) are observed proximally to growth cones that are advancing. Data from 12 independent experiments (pausing, n = 11; advancing, n = 8) are shown as the mean ± SEM. Statistical analysis: unpaired t test. D, Quantification of mitochondrial flux, showing a trend toward more mitochondria arriving versus leaving the growth cone in both pausing and advancing growth cones. Data from 12 independent experiments (pausing, n = 11; advancing, n = 8) are shown as the mean ± SEM. Statistical analysis: unpaired t test. E, Linear regression analysis between the number of mitochondria arriving at a growth cone and growth cone advance (n = 12).

Zebrafish Syntaphilins are expressed in RGCs during development. A, Quantification of snpha and snphb mRNA levels during embryonic development by RT-ddPCR. mRNA levels were normalized to that of gapdh used as a control. Data from three independent experiments are shown as the mean ± SEM. B, Lateral views of whole embryos stained for snphb by ISH show predominant expression in the brain at 48, 72, and 120 hpf. snphb is also increasingly expressed in the RGC layer over time. Scale bars: whole embryos, 400 μm; eyes, 200 μm. C, Dorsal views of WT and RGC-deficient lak mutant embryos stained for snphb by ISH at 72 hpf. Expression of snphb is decreased in the retinae of lak embryos (arrows). Scale bar, 200 μm. D, Quantification of snpha and snphb mRNA levels in the eyes of WT and lak embryos at 72 hpf analyzed by RT-ddPCR. Transcript levels were normalized to that of 18s, which was used as a control. Data from three experiments are shown as the mean ± SEM. Statistical analysis: unpaired t test, ***p < 0.001. E, TagBFP-Snphb localizes to the growth cone of elongating axons in vivo. Isl2b:TagBFP-snphb, isl2b:mitoEGFP-2A-TagRFPCAAX, and isl2b:Lifeact-TagRFP were coexpressed in individual RGCs. TagBFP-Snphb and mitochondria are both present in the growth cone (arrow in merged image). Lateral view, confocal maximal projections. Scale bar, 5 μm.

Zebrafish Syntaphilins anchor mitochondria in mature retinal axons. A, Domain structure of human SNPH. Both the MTB and TMs are highly conserved in zebrafish Snpha and Snphb (see also Extended Data Figure 6-1). The red asterisk indicates the position of the TALEN target region. B, Mutations in snpha and snphb were introduced by TALEN mutagenesis. Red lines indicate the deleted sequences. Changes in amino acids are shown in red. RT-PCR analysis of snpha and snphb in WT and db embryos demonstrate the presence of shorter transcripts in the mutants. C, Individual retinal axons and mitochondria were mosaically labeled by injecting isl2b:mitoEGFP-2A-TagRFPCAAX at the one-cell stage. After removal of the contralateral eye, mature axons of the optic tract and their mitochondria were imaged in a lateral view at 120 hpf (Δt = 15 s). D, E, Quantification of the percentage of stationary mitochondria using kymograph analysis shows a reduction in stalled mitochondria in axons from snph db mutants. Data from two independent experiments (WT: n = 7, db: n = 8) are shown as the mean ± SEM. Statistical analysis: unpaired t test, **p < 0.01.

Syntaphilins participate in mitochondrial docking at the growth cone but do not regulate axon elongation. A–B´, Quantification of the mitochondrial occupancy (as a percentage) of the growth cone total volume (A), total area (Á), central volume (B), and central area (B´) in WT and snph db. Mitochondrial occupancy is decreased in snph db. C, C´, Quantification of the mitochondrial volume in the peripheral growth cone (C), and of the number of mitochondria in the growth cone peripheral area (C´) in WT and snph db. D, D´, Quantification of the distance between the largest mitochondrial cluster and the growth cone leading edge in WT and snph db. We quantified the distance from the leading edge separately in pausing and advancing growth cones as it varies depending on growth cone status (Fig. 2E). Mitochondria are located further from the leading edge in retinal growth cones of db compared with WT. E–G, Reduced mitochondrial occupancy in the growth cone of snph db is accompanied by an increased removal of mitochondria from the growth cone. More mitochondria leave the growth cone per minute in db, while no difference is detected for arriving mitochondria (E). Quantification of net mitochondrial transport proximally to the growth cone (F) shows more mitochondria moving retrogradely in snph db, which is accompanied by a decreased percentage of mitochondria moving in the anterograde direction. No differences in percentage time in anterograde or retrograde motion were found (G). H, I, Quantification of growth cone morphology shows no differences in growth cone total and central areas (H) and number of filopodia (I) between db and WT embryos. J, Axon elongation is not statistically different between db and WT embryos. Data from 12 independent experiments per genotype are shown as the mean ± SEM. Statistical analysis: unpaired t test, **p < 0.01, ***p < 0.001.