Mitochondrial imaging in dorsal root ganglion neurons following the application of inducible adenoviral vector expressing two fluorescent proteins.
Mitochondrial morphology and dynamics are known to vary considerably depending on the cell type and organism studied. The objective of this study was to assess the potential application of adenoviral-fluorescent protein constructs for long-term tracking of mitochondria in neurons. An adenoviral vector containing two fluorescent proteins, the enhanced green fluorescent protein (eGFP) targeted to the cytoplasm to highlight the neuronal processes, and the red fluorescent protein (RFP) directed to mitochondria under the control of an inducible promoter, facilitated an efficient and accurate method to study mitochondrial dynamics in long-term studies. Dorsal root ganglion neurons from rat embryos were cultured and infected. The infected neurons exhibited green fluorescence after 24h, while 16 h following induction with doxycycline, red fluorescence protein began to localize within mitochondria. The red fluorescent protein was transported into mitochondria at the cell body followed by distribution within processes. As the neurons aged, the expression of red fluorescent protein was confined to cytoplasmic vacuoles and not mitochondria. Further analysis suggested that the cytoplasmic vacuoles were likely of lysosomal origin. Taken together, the current study presents novel strategies to study the life history of cellular organelles such as mitochondria in long-term studies.
Department of Biological Sciences, Kent State University, Ashtabula, OH 44004, United States. pnasr@kent.edu
Payman
P
Nasr
Patrick G
PG
Sullivan
George M
GM
Smith
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001370:Axonal Transport / Q000502:physiology; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology; D003593:Cytoplasm / Q000378:metabolism / Q000648:ultrastructure; D004318:Doxycycline / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000166:cytology* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D019044:Image Cytometry / Q000379:methods; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D008247:Lysosomes / Q000378:metabolism / Q000648:ultrastructure; D008856:Microscopy, Fluorescence / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism / Q000648:ultrastructure*; D009475:Neurons, Afferent / Q000166:cytology* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection / Q000379:methods; D014617:Vacuoles / Q000378:metabolism / Q000648:ultrastructure; D000097573:Red Fluorescent Protein
D000900:Anti-Bacterial Agents; D004318:Doxycycline; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein