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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 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.
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fig5: 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.

Mentions: To gain insights into physiological cargoes of this endocytic compartment, we analyzed whether ligands known to undergo retrograde transport in vivo are recruited into TeNT HC carriers. NGF is retrogradely transported by newborn MNs (Yan et al., 1988) and has transport rates similar to TeNT in sensory and adrenergic neurons (Stöckel et al., 1975). In MNs, NGF-containing retrograde carriers could be detected ∼45 min after the end of the incubation at 37°C, consistent with previous studies that used 125I-NGF in sympathetic neurons (Ure and Campenot, 1997). We detected substantial colocalization between TeNT HC and NGF, with 72% of retrograde organelles being double labeled (n = 106 carriers; two independent experiments). These organelles always corresponded to round vesicles (Fig. 5 , a–c; Video 3, available at http://www.jcb.org/cgi/content/full/200106142/DC1). In contrast, we could not observe NGF in retrograde tubules containing TeNT HC (Fig. 5, e–g), suggesting that tubules and round carriers may belong to different retrograde pathways.


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 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.
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getmorefigures.php?uid=PMC2199221&req=5

fig5: 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.
Mentions: To gain insights into physiological cargoes of this endocytic compartment, we analyzed whether ligands known to undergo retrograde transport in vivo are recruited into TeNT HC carriers. NGF is retrogradely transported by newborn MNs (Yan et al., 1988) and has transport rates similar to TeNT in sensory and adrenergic neurons (Stöckel et al., 1975). In MNs, NGF-containing retrograde carriers could be detected ∼45 min after the end of the incubation at 37°C, consistent with previous studies that used 125I-NGF in sympathetic neurons (Ure and Campenot, 1997). We detected substantial colocalization between TeNT HC and NGF, with 72% of retrograde organelles being double labeled (n = 106 carriers; two independent experiments). These organelles always corresponded to round vesicles (Fig. 5 , a–c; Video 3, available at http://www.jcb.org/cgi/content/full/200106142/DC1). In contrast, we could not observe NGF in retrograde tubules containing TeNT HC (Fig. 5, e–g), suggesting that tubules and round carriers may belong to different retrograde pathways.

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