Fluorescence imaging of mitochondria in cultured skin fibroblasts: a useful method for the detection of oxidative phosphorylation defects.
BACKGROUND
Protons are pumped from the mitochondrial matrix via oxidative phosphorylation (OXPHOS) into the intermembrane space, creating an electric membrane potential (ΔΨ) that is used for adenosine triphosphate (ATP) production. Defects in one or more of the OXPHOS complexes are associated with a variety of clinical symptoms, often making it difficult to pinpoint the causal mutation.


METHODS
In this article, a microscopic method for the quantitative evaluation of ΔΨ in cultured skin fibroblasts is described. The method using 5,5',6,6'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) fluorescence staining was tested in a selection of OXPHOS-deficient cell lines.


RESULTS
A significant reduction of ΔΨ was found in the cell lines of patients with either an isolated defect in complex I, II, or IV or a combined defect (complex I + complex IV). ΔΨ was not reduced in the fibroblasts of two patients with severe complex V deficiency. Addition of the complex I inhibitor rotenone induced a significant reduction of ΔΨ and perinuclear relocalization of the mitochondria. In cells with a heteroplasmic mitochondrial DNA (mtDNA) defect, a more heterogeneous reduction of ΔΨ was detected.


CONCLUSIONS
Our data show that imaging of ΔΨ in cultured skin fibroblasts is a useful method for the evaluation of OXPHOS functioning in cultured cell lines.
Department of Pediatrics, Division of Pediatric Neurology and Metabolism, Ghent University Hospital, Ghent, Belgium.
Boel
B
De Paepe
Joél
J
Smet
Arnaud
A
Vanlander
Sara
S
Seneca
Willy
W
Lissens
Linda
L
De Meirleir
Mado
M
Vandewoestyne
Dieter
D
Deforce
Richard J
RJ
Rodenburg
Rudy
R
Van Coster
D002460:Cell Line; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D028361:Mitochondrial Diseases / Q000175:diagnosis*; D012867:Skin / Q000166:cytology / Q000378:metabolism*