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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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Related in: MedlinePlus

Constitutive redistribution of labeled mitochondria.A) Images of a living motor neuron (24 div) were taken within a minute after photo-converting the mitoKaede protein, and then after 18 hours. Over a period of 18 hours mitochondria from the periphery appeared to extensively fuse with organelles from the cell body. Blue circles in the leftmost panels delineate the area of the neuron that was exposed to blue light. Relative fluorescence intensity levels are directly comparable. Scale bars correspond to 20 microns. B) Quantitation of the redistribution of red and green fluorescent mitoKaede. The mean fluorescence intensity per pixel was calculated for both red and green pixels in masks generated solely based on the distribution of red pixels. For each cell (n = 33), the ratio of red: green mean pixel intensity at time 0 h and after 18 h (19±1 h) is plotted as a box and whisker plot. A decrease in this ratio reflects a dilution of the finite amount of red fluorescent mitoKaede with green fluorescent mitoKaede. The median ratio changed from 4.9 to 0.17. C) The level of expression of a short-lived GFP in motor neurons before and after treatment with CHI was quantified by manually generating masks that encompass the green pixels in the cell soma, and calculating the mean fluorescence intensity (n = 10; p<0.001). D) Patch clamp recordings were performed on infected spinal cord neurons sampled from 4 coverslips (n = 7). Spontaneous spiking activity was recorded over 20 s, and action potentials were blocked in all 7 cases by 1 µM TTX. Data are plotted as mean firing rate of the same cells before and after addition of TTX to the ACSF (p<0.001). E) Pooled data showing no significant effect of cycloheximide (CHI 10 µg per ml, n = 20) or tetrodotoxin (TTX, 1 µM, n = 16) on the redistribution of labeled mitochondria over time compared to untreated motor neurons. For each cell in each treatment group, the ratio of red: green mean pixel intensity after 18 h (19±1 h) is normalized to the starting ratio in the same cell, and plotted as a box and whisker plot. F) Box and whisker plots of the change in the size of masks generated from the distribution of red pixels reflecting the dispersion of red fluorescent mitoKaede over time.
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pone-0038435-g002: Constitutive redistribution of labeled mitochondria.A) Images of a living motor neuron (24 div) were taken within a minute after photo-converting the mitoKaede protein, and then after 18 hours. Over a period of 18 hours mitochondria from the periphery appeared to extensively fuse with organelles from the cell body. Blue circles in the leftmost panels delineate the area of the neuron that was exposed to blue light. Relative fluorescence intensity levels are directly comparable. Scale bars correspond to 20 microns. B) Quantitation of the redistribution of red and green fluorescent mitoKaede. The mean fluorescence intensity per pixel was calculated for both red and green pixels in masks generated solely based on the distribution of red pixels. For each cell (n = 33), the ratio of red: green mean pixel intensity at time 0 h and after 18 h (19±1 h) is plotted as a box and whisker plot. A decrease in this ratio reflects a dilution of the finite amount of red fluorescent mitoKaede with green fluorescent mitoKaede. The median ratio changed from 4.9 to 0.17. C) The level of expression of a short-lived GFP in motor neurons before and after treatment with CHI was quantified by manually generating masks that encompass the green pixels in the cell soma, and calculating the mean fluorescence intensity (n = 10; p<0.001). D) Patch clamp recordings were performed on infected spinal cord neurons sampled from 4 coverslips (n = 7). Spontaneous spiking activity was recorded over 20 s, and action potentials were blocked in all 7 cases by 1 µM TTX. Data are plotted as mean firing rate of the same cells before and after addition of TTX to the ACSF (p<0.001). E) Pooled data showing no significant effect of cycloheximide (CHI 10 µg per ml, n = 20) or tetrodotoxin (TTX, 1 µM, n = 16) on the redistribution of labeled mitochondria over time compared to untreated motor neurons. For each cell in each treatment group, the ratio of red: green mean pixel intensity after 18 h (19±1 h) is normalized to the starting ratio in the same cell, and plotted as a box and whisker plot. F) Box and whisker plots of the change in the size of masks generated from the distribution of red pixels reflecting the dispersion of red fluorescent mitoKaede over time.

Mentions: To investigate longer term changes in the distribution of mitochondria, multiple cultures containing photoconverted mitochondria in the cell bodies of several motor neurons were visualized immediately after exposure to blue light and again approximately 18 hours later (19±1 h; n = 33). A Z-stack of images in increments of 0.2 microns, encompassing the entire volume of the cell, was taken at each time point using identical exposure times. As exemplified by the motor neuron shown in Figure 2A, the distribution of green and red fluorescing mitochondria changed over time. After 18 hours the focal area of red fluorescence in the cell body was no longer discernible, and had been replaced by a mixture of red and green fluorescent mitoKaede. Meanwhile the red fluorescence had spread from the cell soma into the neurites. Since the fluorescent protein was targeted to the matrix of the mitochondrion, it appeared that extensive fusion must have taken place between mitochondria from the cell soma and attached neurites in order to account for the observed redistribution of red and green fluorescing proteins. In Figure 2A, the red fluorescence intensity scale was adjusted to the same upper and lower values in both images, and likewise for the green fluorescence intensity scale so that relative expression levels are directly comparable.


Extensive fusion of mitochondria in spinal cord motor neurons.

Owens GC, Walcott EC - PLoS ONE (2012)

Constitutive redistribution of labeled mitochondria.A) Images of a living motor neuron (24 div) were taken within a minute after photo-converting the mitoKaede protein, and then after 18 hours. Over a period of 18 hours mitochondria from the periphery appeared to extensively fuse with organelles from the cell body. Blue circles in the leftmost panels delineate the area of the neuron that was exposed to blue light. Relative fluorescence intensity levels are directly comparable. Scale bars correspond to 20 microns. B) Quantitation of the redistribution of red and green fluorescent mitoKaede. The mean fluorescence intensity per pixel was calculated for both red and green pixels in masks generated solely based on the distribution of red pixels. For each cell (n = 33), the ratio of red: green mean pixel intensity at time 0 h and after 18 h (19±1 h) is plotted as a box and whisker plot. A decrease in this ratio reflects a dilution of the finite amount of red fluorescent mitoKaede with green fluorescent mitoKaede. The median ratio changed from 4.9 to 0.17. C) The level of expression of a short-lived GFP in motor neurons before and after treatment with CHI was quantified by manually generating masks that encompass the green pixels in the cell soma, and calculating the mean fluorescence intensity (n = 10; p<0.001). D) Patch clamp recordings were performed on infected spinal cord neurons sampled from 4 coverslips (n = 7). Spontaneous spiking activity was recorded over 20 s, and action potentials were blocked in all 7 cases by 1 µM TTX. Data are plotted as mean firing rate of the same cells before and after addition of TTX to the ACSF (p<0.001). E) Pooled data showing no significant effect of cycloheximide (CHI 10 µg per ml, n = 20) or tetrodotoxin (TTX, 1 µM, n = 16) on the redistribution of labeled mitochondria over time compared to untreated motor neurons. For each cell in each treatment group, the ratio of red: green mean pixel intensity after 18 h (19±1 h) is normalized to the starting ratio in the same cell, and plotted as a box and whisker plot. F) Box and whisker plots of the change in the size of masks generated from the distribution of red pixels reflecting the dispersion of red fluorescent mitoKaede over time.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368829&req=5

pone-0038435-g002: Constitutive redistribution of labeled mitochondria.A) Images of a living motor neuron (24 div) were taken within a minute after photo-converting the mitoKaede protein, and then after 18 hours. Over a period of 18 hours mitochondria from the periphery appeared to extensively fuse with organelles from the cell body. Blue circles in the leftmost panels delineate the area of the neuron that was exposed to blue light. Relative fluorescence intensity levels are directly comparable. Scale bars correspond to 20 microns. B) Quantitation of the redistribution of red and green fluorescent mitoKaede. The mean fluorescence intensity per pixel was calculated for both red and green pixels in masks generated solely based on the distribution of red pixels. For each cell (n = 33), the ratio of red: green mean pixel intensity at time 0 h and after 18 h (19±1 h) is plotted as a box and whisker plot. A decrease in this ratio reflects a dilution of the finite amount of red fluorescent mitoKaede with green fluorescent mitoKaede. The median ratio changed from 4.9 to 0.17. C) The level of expression of a short-lived GFP in motor neurons before and after treatment with CHI was quantified by manually generating masks that encompass the green pixels in the cell soma, and calculating the mean fluorescence intensity (n = 10; p<0.001). D) Patch clamp recordings were performed on infected spinal cord neurons sampled from 4 coverslips (n = 7). Spontaneous spiking activity was recorded over 20 s, and action potentials were blocked in all 7 cases by 1 µM TTX. Data are plotted as mean firing rate of the same cells before and after addition of TTX to the ACSF (p<0.001). E) Pooled data showing no significant effect of cycloheximide (CHI 10 µg per ml, n = 20) or tetrodotoxin (TTX, 1 µM, n = 16) on the redistribution of labeled mitochondria over time compared to untreated motor neurons. For each cell in each treatment group, the ratio of red: green mean pixel intensity after 18 h (19±1 h) is normalized to the starting ratio in the same cell, and plotted as a box and whisker plot. F) Box and whisker plots of the change in the size of masks generated from the distribution of red pixels reflecting the dispersion of red fluorescent mitoKaede over time.
Mentions: To investigate longer term changes in the distribution of mitochondria, multiple cultures containing photoconverted mitochondria in the cell bodies of several motor neurons were visualized immediately after exposure to blue light and again approximately 18 hours later (19±1 h; n = 33). A Z-stack of images in increments of 0.2 microns, encompassing the entire volume of the cell, was taken at each time point using identical exposure times. As exemplified by the motor neuron shown in Figure 2A, the distribution of green and red fluorescing mitochondria changed over time. After 18 hours the focal area of red fluorescence in the cell body was no longer discernible, and had been replaced by a mixture of red and green fluorescent mitoKaede. Meanwhile the red fluorescence had spread from the cell soma into the neurites. Since the fluorescent protein was targeted to the matrix of the mitochondrion, it appeared that extensive fusion must have taken place between mitochondria from the cell soma and attached neurites in order to account for the observed redistribution of red and green fluorescing proteins. In Figure 2A, the red fluorescence intensity scale was adjusted to the same upper and lower values in both images, and likewise for the green fluorescence intensity scale so that relative expression levels are directly comparable.

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