Limits...
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.

Show MeSH

Related in: MedlinePlus

Characterization of motor neurons in spinal cord cultures.Antibodies were used to confirm the identity of the large neurons growing in the spinal cord cultures (24–29 div). A) A motor neuron (arrow) identified by staining for the non-phosphorylated neurofilament epitope recognized by SMI-32 mAb (green), and a polyclonal antibody against MAP2 (red). The presumptive axon is marked by an asterisk. B) A motor neuron (arrow) visualized with an anti-beta tubulin III antibody (green) does not express the phosphorylated neurofilament epitope recognized by SMI-312 mAb (red), a marker used to identify axons. Nuclei are labeled with DAPI. Scale bars correspond to 50 microns and 20 microns (higher power insets) respectively. C) An infected motor neuron immunostained with SMI-32 mAb (red) showing mitochondria expressing mitoKaede fluorescent protein (green). Arrows point to a close association of mitochondria with neurofilaments. Scale bar corresponds to 20 microns. D) A representative recording of spontaneous activity from an infected spinal cord motor neuron.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3368829&req=5

pone-0038435-g001: Characterization of motor neurons in spinal cord cultures.Antibodies were used to confirm the identity of the large neurons growing in the spinal cord cultures (24–29 div). A) A motor neuron (arrow) identified by staining for the non-phosphorylated neurofilament epitope recognized by SMI-32 mAb (green), and a polyclonal antibody against MAP2 (red). The presumptive axon is marked by an asterisk. B) A motor neuron (arrow) visualized with an anti-beta tubulin III antibody (green) does not express the phosphorylated neurofilament epitope recognized by SMI-312 mAb (red), a marker used to identify axons. Nuclei are labeled with DAPI. Scale bars correspond to 50 microns and 20 microns (higher power insets) respectively. C) An infected motor neuron immunostained with SMI-32 mAb (red) showing mitochondria expressing mitoKaede fluorescent protein (green). Arrows point to a close association of mitochondria with neurofilaments. Scale bar corresponds to 20 microns. D) A representative recording of spontaneous activity from an infected spinal cord motor neuron.

Mentions: Several fluorescent proteins have been developed whose emission maxima are red-shifted following a brief irradiation with blue light [28]. In the present study, a mitochondrial targeting sequence from subunit VIII of cytochrome c oxidase was added to the Kaede protein [27] in order to mark subpopulations of mitochondria in cultured spinal cord motor neurons. Motor neurons were identified by size and morphology, and this was later confirmed by immunocytochemistry (Figure 1A–C). Figure 1D shows a trace of action potentials generated by an infected motor neuron measured by whole cell patch clamp, confirming that infected cells are spontaneously active under the culture conditions used.


Extensive fusion of mitochondria in spinal cord motor neurons.

Owens GC, Walcott EC - PLoS ONE (2012)

Characterization of motor neurons in spinal cord cultures.Antibodies were used to confirm the identity of the large neurons growing in the spinal cord cultures (24–29 div). A) A motor neuron (arrow) identified by staining for the non-phosphorylated neurofilament epitope recognized by SMI-32 mAb (green), and a polyclonal antibody against MAP2 (red). The presumptive axon is marked by an asterisk. B) A motor neuron (arrow) visualized with an anti-beta tubulin III antibody (green) does not express the phosphorylated neurofilament epitope recognized by SMI-312 mAb (red), a marker used to identify axons. Nuclei are labeled with DAPI. Scale bars correspond to 50 microns and 20 microns (higher power insets) respectively. C) An infected motor neuron immunostained with SMI-32 mAb (red) showing mitochondria expressing mitoKaede fluorescent protein (green). Arrows point to a close association of mitochondria with neurofilaments. Scale bar corresponds to 20 microns. D) A representative recording of spontaneous activity from an infected spinal cord motor neuron.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368829&req=5

pone-0038435-g001: Characterization of motor neurons in spinal cord cultures.Antibodies were used to confirm the identity of the large neurons growing in the spinal cord cultures (24–29 div). A) A motor neuron (arrow) identified by staining for the non-phosphorylated neurofilament epitope recognized by SMI-32 mAb (green), and a polyclonal antibody against MAP2 (red). The presumptive axon is marked by an asterisk. B) A motor neuron (arrow) visualized with an anti-beta tubulin III antibody (green) does not express the phosphorylated neurofilament epitope recognized by SMI-312 mAb (red), a marker used to identify axons. Nuclei are labeled with DAPI. Scale bars correspond to 50 microns and 20 microns (higher power insets) respectively. C) An infected motor neuron immunostained with SMI-32 mAb (red) showing mitochondria expressing mitoKaede fluorescent protein (green). Arrows point to a close association of mitochondria with neurofilaments. Scale bar corresponds to 20 microns. D) A representative recording of spontaneous activity from an infected spinal cord motor neuron.
Mentions: Several fluorescent proteins have been developed whose emission maxima are red-shifted following a brief irradiation with blue light [28]. In the present study, a mitochondrial targeting sequence from subunit VIII of cytochrome c oxidase was added to the Kaede protein [27] in order to mark subpopulations of mitochondria in cultured spinal cord motor neurons. Motor neurons were identified by size and morphology, and this was later confirmed by immunocytochemistry (Figure 1A–C). Figure 1D shows a trace of action potentials generated by an infected motor neuron measured by whole cell patch clamp, confirming that infected cells are spontaneously active under the culture conditions used.

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