Figure 3.
Figure 3. Autophagosomes acquire retrograde motility by fusion with LEs. (A and B) Stx17 knockdown blocks formation of amphisomes. (A) Imaging was performed on the middle to distal segments of axons. Arrowheads indicate amphisomes (yellow) colabeled with LC3 and Rab7, whereas arrows indicate autophagosomes (green) without Rab7 labeling. Note a robust increase in autophagosomes and decrease in amphisomes in axons expressing Stx17-siRNA (B). The number of autophagosomes or amphisomes was expressed as a percentage of the total LC3-labeled AVs. Data were quantified from 28 axons in each group and a total number of 293 AVs in greater than three experiments. (C–E) Relative motility of amphisomes, autophagosomes, and LEs. (C, right) Note that in neurons with Stx17 knockdown, autophagosomes (green) were largely stationary, whereas LEs (red) underwent predominant retrograde transport, some of which passed through stationary autophagosomes. (C and E) Depleting Stx17 had no effect on the motility of LEs. Quantification was performed from the total number of AVs or LEs (V, vesicles) from the total number of neurons (N) in greater than three experiments. (F and G) TEM analysis showing early stage AVi versus late-stage AVd in DRG neurons. In Stx17 knockdown neurites, the majority of AV-like structures are early stage autophagosomes (AVi; green box and arrows) and were easily found. In control neurons, the majority of AVs are late-stage autophagic vacuoles (AVd) containing electron-dense material and small vesicles (orange box and arrows), suggesting fusion with LEs. Data were collected from 61 micrographs (5 × 5 µm) for each condition. (H) Dynamic de novo autophagosomal biogenesis, fusion, and retrograde transport in a DRG neuron growth cone. Time-lapse imaging was taken once every 3 s with acquisition exposure time at 150 ms in a Nikon spinning-disk confocal. White arrows point to the appearance of new autophagosomes within growth cone at 15, 54, and 63 s; the yellow arrow denotes fusion events, and yellow arrowheads indicate a retrograde motile amphisome between 33 and 87 s (also see Video 1). Error bars: SEM. Unpaired Student’s t test. Bars: (A and H) 5 µm; (C) 10 µm; (F, main images) 200 nm; (F, boxes) 100 nm.

Autophagosomes acquire retrograde motility by fusion with LEs. (A and B) Stx17 knockdown blocks formation of amphisomes. (A) Imaging was performed on the middle to distal segments of axons. Arrowheads indicate amphisomes (yellow) colabeled with LC3 and Rab7, whereas arrows indicate autophagosomes (green) without Rab7 labeling. Note a robust increase in autophagosomes and decrease in amphisomes in axons expressing Stx17-siRNA (B). The number of autophagosomes or amphisomes was expressed as a percentage of the total LC3-labeled AVs. Data were quantified from 28 axons in each group and a total number of 293 AVs in greater than three experiments. (C–E) Relative motility of amphisomes, autophagosomes, and LEs. (C, right) Note that in neurons with Stx17 knockdown, autophagosomes (green) were largely stationary, whereas LEs (red) underwent predominant retrograde transport, some of which passed through stationary autophagosomes. (C and E) Depleting Stx17 had no effect on the motility of LEs. Quantification was performed from the total number of AVs or LEs (V, vesicles) from the total number of neurons (N) in greater than three experiments. (F and G) TEM analysis showing early stage AVi versus late-stage AVd in DRG neurons. In Stx17 knockdown neurites, the majority of AV-like structures are early stage autophagosomes (AVi; green box and arrows) and were easily found. In control neurons, the majority of AVs are late-stage autophagic vacuoles (AVd) containing electron-dense material and small vesicles (orange box and arrows), suggesting fusion with LEs. Data were collected from 61 micrographs (5 × 5 µm) for each condition. (H) Dynamic de novo autophagosomal biogenesis, fusion, and retrograde transport in a DRG neuron growth cone. Time-lapse imaging was taken once every 3 s with acquisition exposure time at 150 ms in a Nikon spinning-disk confocal. White arrows point to the appearance of new autophagosomes within growth cone at 15, 54, and 63 s; the yellow arrow denotes fusion events, and yellow arrowheads indicate a retrograde motile amphisome between 33 and 87 s (also see Video 1). Error bars: SEM. Unpaired Student’s t test. Bars: (A and H) 5 µm; (C) 10 µm; (F, main images) 200 nm; (F, boxes) 100 nm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal