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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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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.
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fig3: 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.

Mentions: Conflicting results on the fate of TeNT after endocytosis have been reported. Although some suggested that TeNT might escape lysosomal degradation in vivo, others found TeNT in multivesicular bodies and lysosomes (Schiavo et al., 2000). To characterize the TeNT HC compartment, we performed two-color time-lapse microscopy in living MNs using the membrane-permeable dye Lysotracker, which stains acidic organelles and lysosomes. We observed no colocalization between TeNT HC–labeled endosomes and Lysotracker-stained vesicles (Fig. 3 , a–c; Video 2, available at http://www.jcb.org/cgi/content/full/200106142/DC1). Confocal time-lapse experiments and simultaneous differential interference contrast (DIC) imaging revealed that Lysotracker was particularly concentrated in phase-contrast bright round structures (Fig. 3, d–e, *), which are likely to correspond to prelysosomal organelles (Kuznetsov et al., 1992) and were always distinct from the round or tubulo-vesicular carriers labeled by TeNT HC and undetectable by DIC (Fig. 3, d–e). The Lysotracker-positive compartment is accessible to the endocytic tracer Texas red dextran (Fig. 3, f–h). Notably, we observed organelles stained by fluorescent dextran, which were not acidic (Fig. 3 h, *).


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)

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.
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fig3: 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.
Mentions: Conflicting results on the fate of TeNT after endocytosis have been reported. Although some suggested that TeNT might escape lysosomal degradation in vivo, others found TeNT in multivesicular bodies and lysosomes (Schiavo et al., 2000). To characterize the TeNT HC compartment, we performed two-color time-lapse microscopy in living MNs using the membrane-permeable dye Lysotracker, which stains acidic organelles and lysosomes. We observed no colocalization between TeNT HC–labeled endosomes and Lysotracker-stained vesicles (Fig. 3 , a–c; Video 2, available at http://www.jcb.org/cgi/content/full/200106142/DC1). Confocal time-lapse experiments and simultaneous differential interference contrast (DIC) imaging revealed that Lysotracker was particularly concentrated in phase-contrast bright round structures (Fig. 3, d–e, *), which are likely to correspond to prelysosomal organelles (Kuznetsov et al., 1992) and were always distinct from the round or tubulo-vesicular carriers labeled by TeNT HC and undetectable by DIC (Fig. 3, d–e). The Lysotracker-positive compartment is accessible to the endocytic tracer Texas red dextran (Fig. 3, f–h). Notably, we observed organelles stained by fluorescent dextran, which were not acidic (Fig. 3 h, *).

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