Do single mitochondria contain zones with different membrane potential?

Abstract

Observations of Lan Bo Chen’s group using a mitochondria-selective fluorochrome 5,5’,6,6’- tetrachloro- 1,1’,3,3’- tetraethylbenzimidazolocarbocyanine iodide (JC-1) indicate that mitochondria in situ may have zones of different electrochemical potential along their length. This was indicated by the formation of J-aggregates of this dye at distinct sites along a single mitochondrion. Also, intensity variations along single mitochondria were found with diamino-styryl-pyridinium methiodide (DASPMI), another fluorochrome that selectively stains mitochondria depending on their electrochemical potential. DASPMI exchanges easily with the cytoplasm and changes its quantum yield when bound to mitochondrial membranes. Therefore, fluorescence intensity is primarily controlled by the membrane environment rather than by mass accumulation. Two possible explanations of intramitochondrial fluorescence intensity variations have to be discussed: variations in the amount of mitochondrial inner membrane per unit of projection area (or voxel), and differences in the electrochemical gradient. This problem has been approached by comparing fluoro-micrographs of mitochondria in endothelial cells stained with either JC-1 or DASPMI with electron micrographs of the same mitochondria after fixation with glutardialdehyde and osmium tetroxide and ultrathin sectioning. JC-1 red fluorescence (revealing J-aggregate formation) as well as high-intensity staining with DASPMI correlate roughly with the local thickness of mitochondria; no differences in the crista organization are revealed for those areas or mitochondria exhibiting red JC-1 fluorescence and those with green fluorescence. The distance between red fluorescing areas in a single mitochondrion seem to be caused by competition for dye molecules placed in between centres of JC-1 aggregation. Isolated mitochondria are of uniform small size and spherical shape; therefore, no differences in shape interfere with JC-1 staining. Thus JC-1 may be an appropriate indicator of membrane potential in isolated mitochondria. In living cells mitochondria often are large and elongated, and thus the situation is not straightforward to interpret. However, evidence is provided that there are submitochondrial zones, which differ in membrane potential from one adjacent area to another, because DASPMI staining of intramitochondrial zones reveals differences in fluorescence intensity and preferred photodamage of these areas. In some cases separation of the zones of higher membrane potential by cristae traversing the whole diameter of a mitochondrion has been observed. Local photobleaching of stained mitochondria results in a loss of fluorescence along the total length of a mitochondrion. However, this type of bleaching develops over tens of seconds, not in the very short time range (e.g. ms) expected from the discharge of all the membranes if they were electrically coupled.