Analysis of retrograde transport in motor neurons reveals common endocytic carriers for tetanus toxin and neurotrophin receptor p75NTR

Abstract

Axonal retrograde transport is essential for neuronal growth and survival. However, the nature and dynamics of the membrane compartments involved in this process are poorly characterized. To shed light on this pathway, we established an experimental system for the visualization and the quantitative study of retrograde transport in living motor neurons based on a fluorescent fragment of tetanus toxin (TeNT HC). Morphological and kinetic analysis of TeNT HC retrograde carriers reveals two major groups of organelles: round vesicles and fast tubular structures. TeNT HC carriers lack markers of the classical endocytic pathway and are not acidified during axonal transport. Importantly, TeNT HC and NGF share the same retrograde transport organelles, which are characterized by the presence of the neurotrophin receptor p75NTR. Our results provide the first direct visualization of retrograde transport in living motor neurons, and reveal a novel retrograde route that could be used both by physiological ligands (i.e., neurotrophins) and TeNT to enter the central nervous system.

Figures

Figure 1.

Visualization of TeNT HC retrograde carriers. Cells were incubated with TeNT HC-Alexa488 for 15 min at 37°C, washed, and imaged by low-light time-lapse microscopy. (a) Low-magnification image of an MN displaying vesicular staining. (b–g) Time series imaging of the axon in the boxed area in a. Intervals between frames are 5 s. Both round vesicles (arrow) and tubular structures (arrowhead) travel toward the cell body (see Video 1, available at http://www.jcb.org/cgi/content/full/200106142/DC1). (h–j) Example of a tubular endosome bending during retrograde transport along a single axon (arrowhead). A slower round vesicle is also indicated (asterisk). The cell body is located out of view at the bottom of the picture. Bars: (a–g) 5 μm; (h–j) 2 μm.

Figure 2.

Kinetic analysis of TeNT HC carriers in MNs. (a) Displacement of 15 carriers tracked during a representative experiment. Retrograde movement is conventionally shown as positive. Intervals between time points are 5 s. Note the presence of fast tubules (•) and slower vesicles (□). Half-filled squares refer to other types of carriers. (b) Displacement graphs of the tubules from a, setting the start of tracking as time 0, show an apparently constant movement with similar speed. (c) Displacement graphs of the remaining carriers from a show a slower and more discontinuous movement. (d) Average speeds of TeNT HC retrograde carriers (▵; n = 256 carriers from three independent experiments). Round vesicles (□; n = 76) have an average speed distribution different from tubules (•, n = 93). (e) Percentage of the different types of carriers analyzed in d.

Figure 3.

TeNT HC carriers do not colocalize with acidic organelles. MNs were incubated with TeNT HC Alexa488 and Lysotracker red DND-99 for 20 min at 37°C. Cells were then washed and imaged with low-light microscopy. The cell body is located out of view to the right. Intervals between frames are 5 s. (a) Time series showing retrograde TeNT HC–labeled endosomes (arrow and •). (b) Corresponding frames showing Lysotracker-stained organelles (arrowheads). (c) Merged images of a and b. Note the lack of colocalization between TeNT HC and Lysotracker-stained organelles (see Video 2, available at http://www.jcb.org/cgi/content/full/200106142/DC1). (d–e) Detail from confocal observation of an axonal branch point. (d) DIC image. (e) Overlap of the green and red channels with the simultaneous DIC image. TeNT HC (green) stains tubular and round carriers (arrows), whereas Lysotracker (red) labels distinct round vesicles (* and arrowheads). An asterisk marks a phase-contrast bright round organelle positive for Lysotracker, but negative for TeNT HC. (f–h) Lysotracker-positive organelles are accessible to endocytic tracers. MNs were incubated with Texas red dextran overnight and with Lysotracker green DND-26 for 30 min at 37°C. Cells were then washed and imaged by confocal microscopy. Lysotracker-positive organelles (f) are also stained by Texas red dextran (g, arrowheads). (h) Merged image of f and g. Nonacidic organelles containing only dextran are also visible (*). Bars, 5 μm.

Figure 4.

TeNT HC carriers and Lysotracker-positive organelles show distinct motile properties. (a) Relative frequency of speed values observed between two consecutive frames (interval = 5 s) for TeNT HC carriers (n = 364 vesicles, black bars) and Lysotracker-containing organelles (n = 235 vesicles, gray bars). Retrograde movement is conventionally shown as positive. (b) The incidence of reversal (number of changes of direction per organelle) for Lysotracker-positive vesicles is 11 times higher than TeNT HC carriers.

Figure 5.

TeNT HC retrograde carriers partially colocalize with NGF-labeled compartments. MNs were incubated with TeNT HC Alexa488 and Texas red NGF for 30 min at 37°C. Cells were then washed and imaged by time-lapse confocal microscopy. The cell body is located out of view to the right. (a) TeNT HC Alexa488 and (b) Texas red NGF colocalize in retrograde carriers (arrowhead, asterisk, arrow) (see Video 3, available at http://www.jcb.org/cgi/content/full/200106142/DC1). (c) Merged image of a and b. (d) Corresponding DIC image. TeNT HC (e) and NGF (f) are found only in round vesicles (arrowhead), whereas tubules appear to be labeled only by TeNT HC (*). (g) Merged image of e and f. (h–j) Many TeNT HC carriers colocalize with p75NTR (arrowheads). MNs were incubated with TeNT HC Alexa488 (h), fixed, immunostained for p75NTR (i), and imaged by confocal microscopy. (j) Merged image of h and i. Bars, 5 μm.