Fluorescence Imaging Of The Mitochondrial Peroxynitrite

The mitochondrion is one of the essential organelles in most eukaryotes.Its functions involve energy supply,signal transduction,cell differentiation,cell death and maintenance of cell cycle and growth control.Any damage and dysfunction in mitochondria may be the key factors leading to a series of human diseases.It has been reported that abnormal changes of mitochondrial membrane potential are important factors leading to mitochondrion dysfunction and mitochondrion related diseases.Peroxynitrite(ONOO~-)is a kind of highly active oxygen species.It has strong cytotoxicity,can damage the structure and function of mitochondria,and can significantly reduce or even completely eliminate the mitochondrial membrane potential.Therefore,it is particularly important to detect the concentration of ONOO~-in mitochondria.The traditional mitochondrion-targeted fluorescent probes own positive charges in their structures,which are attracted into mitochondria due to the negative potential of the mitochondrial inner membrane.Using the traditional mitochondrial targeting strategy,we integrate the formamide group which selectively responses to ONOO~-into the naphthalimide fluorophore,and connect it with pyridine cation group to construct the mitochondrion-targeted fluorescent probe Mito T.Mito T reacts with ONOO~-,causing the cleavage of the amide bond,and the release of naphthalimide fluorophore and the yellowish green fluorescence.With fast response,high sensitivity and good selectivity,the probe can perform two-photon fluorescence imaging of ONOO~-in mitochondria of living cells.However,ONOO~-has strong cytotoxicity,which can damage the structure and function of mitochondria,significantly reduce or even completely eliminate the membrane potential,and correspondingly reduce the affinity of mitochondria to the probe,thus interfere with the targeting ability of the probe to mitochondria.Therefore,Mito T is unable to detect the mitochondrial ONOO~-stably and accurately.Based on the above results,a novel mitochondrion targeted and anchored fluorescent probe,Mito A,is designed and synthesized.Mito A employs naphthalimide as the fluorescence signal unit and formamide as the ONOO~-responsive group,and pyridine cation and benzyl chloride groups are introduced into the probe structure as the mitochondrial targeting group and the anchoring group respectively.Mito A has good selectivity,high sensitivity and fast response to ONOO~-and its response performance is basically the same as that of Mito T.Compared with Mito T,the advantage of Mito A is that the benzyl chloride group in the structure can be covalently linked with mitochondrial sulfhydryl protein,making it anchored in mitochondria,effectively solving the problem of probe leakage from mitochondria caused by the decrease of membrane potential,so as to carry out stable and accurate fluorescence imaging detection of ONOO~-in mitochondria.With the aid of Mito A,the fluorescence imaging shows that cells produce ONOO~-continuously during the starvation induces autophagy,which reveals the close relationship between autophagy and ONOO~-.This study provides a new fluorescent probe for anchoring mitochondria and imaging ONOO~-in mitochondria,which effectively eliminates the interference of mitochondrial membrane potential changes on probe imaging,and can stably and accurately detect the concentration of ONOO~-in autophagy and other pathological processes.