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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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Inhibition of neuronal activity with TTX does not affect mitochondrial fusion in neurites.Kymographs show that fusion of mitochondria was unaffected by continuously blocking neuronal activity with TTX (1 µM final concentration). Time lapse images were taken every 10 seconds for 30 minutes at 0 h, 4 h, and 18 h following the marking of a subpopulation of mitochondria within a living neurite (27 div).
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pone-0038435-g005: Inhibition of neuronal activity with TTX does not affect mitochondrial fusion in neurites.Kymographs show that fusion of mitochondria was unaffected by continuously blocking neuronal activity with TTX (1 µM final concentration). Time lapse images were taken every 10 seconds for 30 minutes at 0 h, 4 h, and 18 h following the marking of a subpopulation of mitochondria within a living neurite (27 div).

Mentions: In the course of imaging individual neurites, fusion events were captured in time lapse images taken in the first 30 minutes following exposure to blue light. Portions of two time lapse series are shown in Movies S2 and S3. In Movie S2, a slow moving mitochondrion is seen to fuse with a second mitochondrion approximately 10 microns away; in Movie S3, a highly motile organelle enters the field of view and fuses with a stationary organelle. To visualize both rapid movement and fusion over time, images of photoconverted mitochondria in a length of neurite were acquired every 10 seconds for 30 minutes and then again after 5 hours, and 21 hours. The time lapse images were plotted as kymographs (Figure 4) and were also converted into QuickTime movies (Movie S4, Movie S5, and Movie S6). By 5 hours, fusion of red fluorescing mitochondria with green fluorescing mitochondria entering from both the proximal and distal directions was clearly evident, and after 21 hours the entire field of view was occupied by mitochondria containing both red and green fluorescent protein. Superimposed upon this process, moving mitochondria were apparent at all time points traversing the approximately 140 microns of neurite under observation. In the first 5 hours following irradiation motile red and green fluorescing mitochondria were discernible; by 21 hours moving mitochondria co-expressed both red and green fluorescent protein, indicating that they were the product of a fusion event that had occurred in the intervening time. Adding TTX did not appear to alter the fusion of mitochondria within neurites (Figure 5). Particle tracking was used to determine the velocity of moving mitochondria, including the mitochondria that were observed to fuse (Movies S2 and S3). Measurements were made of individual mitochondria that moved without stopping in consecutive images taken from 12 independent time lapse series. The extent of continuous movement, either towards or away from the cell body, varied from only 5 microns to 58.9 microns and the average speed was 0.19±0.1 microns per second (n = 14). Maximum velocities ranged from 0.16 to 0.79 microns per second.


Extensive fusion of mitochondria in spinal cord motor neurons.

Owens GC, Walcott EC - PLoS ONE (2012)

Inhibition of neuronal activity with TTX does not affect mitochondrial fusion in neurites.Kymographs show that fusion of mitochondria was unaffected by continuously blocking neuronal activity with TTX (1 µM final concentration). Time lapse images were taken every 10 seconds for 30 minutes at 0 h, 4 h, and 18 h following the marking of a subpopulation of mitochondria within a living neurite (27 div).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368829&req=5

pone-0038435-g005: Inhibition of neuronal activity with TTX does not affect mitochondrial fusion in neurites.Kymographs show that fusion of mitochondria was unaffected by continuously blocking neuronal activity with TTX (1 µM final concentration). Time lapse images were taken every 10 seconds for 30 minutes at 0 h, 4 h, and 18 h following the marking of a subpopulation of mitochondria within a living neurite (27 div).
Mentions: In the course of imaging individual neurites, fusion events were captured in time lapse images taken in the first 30 minutes following exposure to blue light. Portions of two time lapse series are shown in Movies S2 and S3. In Movie S2, a slow moving mitochondrion is seen to fuse with a second mitochondrion approximately 10 microns away; in Movie S3, a highly motile organelle enters the field of view and fuses with a stationary organelle. To visualize both rapid movement and fusion over time, images of photoconverted mitochondria in a length of neurite were acquired every 10 seconds for 30 minutes and then again after 5 hours, and 21 hours. The time lapse images were plotted as kymographs (Figure 4) and were also converted into QuickTime movies (Movie S4, Movie S5, and Movie S6). By 5 hours, fusion of red fluorescing mitochondria with green fluorescing mitochondria entering from both the proximal and distal directions was clearly evident, and after 21 hours the entire field of view was occupied by mitochondria containing both red and green fluorescent protein. Superimposed upon this process, moving mitochondria were apparent at all time points traversing the approximately 140 microns of neurite under observation. In the first 5 hours following irradiation motile red and green fluorescing mitochondria were discernible; by 21 hours moving mitochondria co-expressed both red and green fluorescent protein, indicating that they were the product of a fusion event that had occurred in the intervening time. Adding TTX did not appear to alter the fusion of mitochondria within neurites (Figure 5). Particle tracking was used to determine the velocity of moving mitochondria, including the mitochondria that were observed to fuse (Movies S2 and S3). Measurements were made of individual mitochondria that moved without stopping in consecutive images taken from 12 independent time lapse series. The extent of continuous movement, either towards or away from the cell body, varied from only 5 microns to 58.9 microns and the average speed was 0.19±0.1 microns per second (n = 14). Maximum velocities ranged from 0.16 to 0.79 microns per second.

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
Related in: MedlinePlus