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Extensive fusion of mitochondria in spinal cord motor neurons.

Owens GC, Walcott EC - PLoS ONE (2012)

Bottom Line: The behavior of these selectively labeled mitochondria was followed by live fluorescence imaging.Marking mitochondria within the cell soma revealed a complete mixing, within 18 hours, of these organelles with mitochondria coming from the surrounding neurites.Within 24 hours, photoconverted mitoKaede was dispersed to all of the mitochondria in the portion of neurite under observation.

View Article: PubMed Central - PubMed

Affiliation: The Neurosciences Institute, San Diego, California, United States of America. owens@nsi.edu

ABSTRACT
The relative roles played by trafficking, fission and fusion in the dynamics of mitochondria in neurons have not been fully elucidated. In the present study, a slow widespread redistribution of mitochondria within cultured spinal cord motor neurons was observed as a result of extensive organelle fusion. Mitochondria were labeled with a photoconvertible fluorescent protein (mitoKaede) that is red-shifted following brief irradiation with blue light. The behavior of these selectively labeled mitochondria was followed by live fluorescence imaging. Marking mitochondria within the cell soma revealed a complete mixing, within 18 hours, of these organelles with mitochondria coming from the surrounding neurites. Fusion of juxtaposed mitochondria was directly observed in neuritic processes at least 200 microns from the cell body. Within 24 hours, photoconverted mitoKaede was dispersed to all of the mitochondria in the portion of neurite under observation. When time lapse imaging over minutes was combined with long-term observation of marked mitochondria, moving organelles that traversed the field of view did not initially contain photoconverted protein, but after several hours organelles in motion contained both fluorescent proteins, coincident with widespread fusion of all of the mitochondria within the length of neurite under observation. These observations suggest that there is a widespread exchange of mitochondrial components throughout a neuron as a result of organelle fusion.

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Time course of the fusion of individual mitochondria.A living neurite (28 div) with mitochondria containing photoconverted mitoKaede was imaged over several hours. The appearance of green fluorescing mitoKaede within individual red fluorescing mitochondria (labeled i- iii) during 4 hours of observation is seen. The arrow indicates the location of a mitochondrion that had entered the field of view before the first image was taken. By 20 hours, the red fluorescent protein was distributed throughout the mitochondria under observation. Scale bars correspond to 5 microns.
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pone-0038435-g003: Time course of the fusion of individual mitochondria.A living neurite (28 div) with mitochondria containing photoconverted mitoKaede was imaged over several hours. The appearance of green fluorescing mitoKaede within individual red fluorescing mitochondria (labeled i- iii) during 4 hours of observation is seen. The arrow indicates the location of a mitochondrion that had entered the field of view before the first image was taken. By 20 hours, the red fluorescent protein was distributed throughout the mitochondria under observation. Scale bars correspond to 5 microns.

Mentions: The reproducible change in the pattern of red and green mitoKaede expression strongly implied that widespread fusion between organelles from the soma and neurites takes place leading to a mixing of their contents over time. The limits of far field fluorescence microscopy employed in this study precluded definitively resolving individual mitochondria that had undergone fusion within the cell body of a motor neuron. On the other hand it was possible to follow the fusion of individual organelles within surrounding neurites. A circle ∼30 microns in diameter, and at least 200 microns from an identified cell body, was irradiated with blue light to mark mitochondria in a single neuritic process. Spinal cord motor neurons elaborate multiple branched dendrites and a single long axon, which may form collaterals [29], [30], and as shown in Figure 1 the cultured motor neurons used in this study were highly polarized. However, in living cultures expressing mitoKaede it was not possible to unequivocally distinguish the axon from long dendrites, but given the greater prevalence of the latter, it was more likely that a given neurite under observation was a dendrite. Fluorescence images were taken every hour for four hours and then again after 20 hours. As exemplified in Figure 3 there was clear evidence of fusion between juxtaposed mitochondria over time. The fate of individual mitochondria could be followed over the 4 hour period (mitochondria are labeled i-iii in Figure 3). Between the time of blue light irradiation and image acquisition, a single green mitochondrion had moved into the territory occupied by the red mitochondria but had not fused (arrow). The amount of red fluorescent protein in mitochondria in the photoconverted area was reduced after 20 hours, whereas all of the organelles in the field of view contained much more green fluorescent protein (Figure 3). This implied that the contents of the original red fluorescing mitochondria had been dispersed by multiple fusion events with green fluorescing mitochondria coming from outside the region under observation. At the same time the photoconverted protein had spread to mitochondria away from the original site of photoconversion.


Extensive fusion of mitochondria in spinal cord motor neurons.

Owens GC, Walcott EC - PLoS ONE (2012)

Time course of the fusion of individual mitochondria.A living neurite (28 div) with mitochondria containing photoconverted mitoKaede was imaged over several hours. The appearance of green fluorescing mitoKaede within individual red fluorescing mitochondria (labeled i- iii) during 4 hours of observation is seen. The arrow indicates the location of a mitochondrion that had entered the field of view before the first image was taken. By 20 hours, the red fluorescent protein was distributed throughout the mitochondria under observation. Scale bars correspond to 5 microns.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368829&req=5

pone-0038435-g003: Time course of the fusion of individual mitochondria.A living neurite (28 div) with mitochondria containing photoconverted mitoKaede was imaged over several hours. The appearance of green fluorescing mitoKaede within individual red fluorescing mitochondria (labeled i- iii) during 4 hours of observation is seen. The arrow indicates the location of a mitochondrion that had entered the field of view before the first image was taken. By 20 hours, the red fluorescent protein was distributed throughout the mitochondria under observation. Scale bars correspond to 5 microns.
Mentions: The reproducible change in the pattern of red and green mitoKaede expression strongly implied that widespread fusion between organelles from the soma and neurites takes place leading to a mixing of their contents over time. The limits of far field fluorescence microscopy employed in this study precluded definitively resolving individual mitochondria that had undergone fusion within the cell body of a motor neuron. On the other hand it was possible to follow the fusion of individual organelles within surrounding neurites. A circle ∼30 microns in diameter, and at least 200 microns from an identified cell body, was irradiated with blue light to mark mitochondria in a single neuritic process. Spinal cord motor neurons elaborate multiple branched dendrites and a single long axon, which may form collaterals [29], [30], and as shown in Figure 1 the cultured motor neurons used in this study were highly polarized. However, in living cultures expressing mitoKaede it was not possible to unequivocally distinguish the axon from long dendrites, but given the greater prevalence of the latter, it was more likely that a given neurite under observation was a dendrite. Fluorescence images were taken every hour for four hours and then again after 20 hours. As exemplified in Figure 3 there was clear evidence of fusion between juxtaposed mitochondria over time. The fate of individual mitochondria could be followed over the 4 hour period (mitochondria are labeled i-iii in Figure 3). Between the time of blue light irradiation and image acquisition, a single green mitochondrion had moved into the territory occupied by the red mitochondria but had not fused (arrow). The amount of red fluorescent protein in mitochondria in the photoconverted area was reduced after 20 hours, whereas all of the organelles in the field of view contained much more green fluorescent protein (Figure 3). This implied that the contents of the original red fluorescing mitochondria had been dispersed by multiple fusion events with green fluorescing mitochondria coming from outside the region under observation. At the same time the photoconverted protein had spread to mitochondria away from the original site of photoconversion.

Bottom Line: The behavior of these selectively labeled mitochondria was followed by live fluorescence imaging.Marking mitochondria within the cell soma revealed a complete mixing, within 18 hours, of these organelles with mitochondria coming from the surrounding neurites.Within 24 hours, photoconverted mitoKaede was dispersed to all of the mitochondria in the portion of neurite under observation.

View Article: PubMed Central - PubMed

Affiliation: The Neurosciences Institute, San Diego, California, United States of America. owens@nsi.edu

ABSTRACT
The relative roles played by trafficking, fission and fusion in the dynamics of mitochondria in neurons have not been fully elucidated. In the present study, a slow widespread redistribution of mitochondria within cultured spinal cord motor neurons was observed as a result of extensive organelle fusion. Mitochondria were labeled with a photoconvertible fluorescent protein (mitoKaede) that is red-shifted following brief irradiation with blue light. The behavior of these selectively labeled mitochondria was followed by live fluorescence imaging. Marking mitochondria within the cell soma revealed a complete mixing, within 18 hours, of these organelles with mitochondria coming from the surrounding neurites. Fusion of juxtaposed mitochondria was directly observed in neuritic processes at least 200 microns from the cell body. Within 24 hours, photoconverted mitoKaede was dispersed to all of the mitochondria in the portion of neurite under observation. When time lapse imaging over minutes was combined with long-term observation of marked mitochondria, moving organelles that traversed the field of view did not initially contain photoconverted protein, but after several hours organelles in motion contained both fluorescent proteins, coincident with widespread fusion of all of the mitochondria within the length of neurite under observation. These observations suggest that there is a widespread exchange of mitochondrial components throughout a neuron as a result of organelle fusion.

Show MeSH