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

Lalli G, Schiavo G - J. Cell Biol. (2002)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Molecular NeuroPathoBiology Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom.

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.

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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.
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fig1: 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.

Mentions: We used TeNT HC Alexa488 to follow retrograde transport in living MNs. Cells were incubated with 25–40 nM fluorescent TeNT HC for 15 min at 37°C, washed, and imaged by confocal or low-light microscopy. The plasma membrane appeared highly stained, particularly at neurite contacts and synaptic sites. Cell bodies and dendrites also displayed a bright staining pattern (Fig. 1 a), suggesting that TeNT HC binding sites are not restricted to the axonal surface in cultured MNs. An earlier study suggested that TeNT could enter hippocampal neurons through synaptic vesicle recycling (Matteoli et al., 1996). In contrast, in MNs, TeNT HC is internalized in the absence of depolarization and displays very limited colocalization with synaptic vesicle markers (unpublished data). This is in agreement with reports showing that TeNT uptake and retrograde transport are unaffected at NMJs in which neurotransmitter release has been inhibited (Schiavo et al., 2000). Therefore, the uptake mechanism of TeNT at the NMJ could be different from that occurring in central neurons.


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

Lalli G, Schiavo G - J. Cell Biol. (2002)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199221&req=5

fig1: 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.
Mentions: We used TeNT HC Alexa488 to follow retrograde transport in living MNs. Cells were incubated with 25–40 nM fluorescent TeNT HC for 15 min at 37°C, washed, and imaged by confocal or low-light microscopy. The plasma membrane appeared highly stained, particularly at neurite contacts and synaptic sites. Cell bodies and dendrites also displayed a bright staining pattern (Fig. 1 a), suggesting that TeNT HC binding sites are not restricted to the axonal surface in cultured MNs. An earlier study suggested that TeNT could enter hippocampal neurons through synaptic vesicle recycling (Matteoli et al., 1996). In contrast, in MNs, TeNT HC is internalized in the absence of depolarization and displays very limited colocalization with synaptic vesicle markers (unpublished data). This is in agreement with reports showing that TeNT uptake and retrograde transport are unaffected at NMJs in which neurotransmitter release has been inhibited (Schiavo et al., 2000). Therefore, the uptake mechanism of TeNT at the NMJ could be different from that occurring in central neurons.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Molecular NeuroPathoBiology Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus