Use of nonyl acridine orange and rhodamine 123 to follow biosynthesis and functional assembly of mitochondrial membrane during L1210 cell cycle

Abstract

Specific mitochondrial incorporation of 10 N-nonyl acridine orange (NAO) is demonstrated by subcellular fractionation of rat hepatocytes. Moreover, comparative studies with NAO and rhodamine 123 (Rh 123) prove that acridine orange-derivative uptake is independent of transmembrane mitochondrial potential, a property allowing its utilization for the assessment of mitochondrial membrane mass modifications under various physiological states. Using NAO and Rh 123, we have respectively followed the biosynthesis of mitochondrial membrane and its assembly under a functional state during the L1210 cell cycle. Their evolution occurs in two stages according to a well-defined sequential order. Mitochondrial biogenesis, as revealed by NAO incorporation, occurs essentially in the G1 phase (probably mitochondrion enlargement) but also starts in late S phase (probably mitochondrion division). The increased amount of functional mitochondrial membrane, monitored by Rh 123 uptake, is emphasized in late G1 (prerequisite to DNA synthesis) and during G2M phases (prerequisite to mitosis). This alternative succession of phases displays the existence of a time-lag between the biosynthesis of mitochondrial membrane and its functional organization. Such an analysis confirms the potential of the NAO probe to evaluate mitochondrial membrane mass changes in various biological fields.