Inner Membrane Protein Of Mitochondria (IMMT)

As highly dynamic double membrane bound organelles in cells, mitochondria exhibit common structural features in general but various cristae shape due to different energy demand and metabolic states, which is derived from the infolded inner membrane where protein complexes of oxidative phosphorylation and intermediate metabolism are embedded to produce ATP for the basic life activity. Abundant and complex cristae are found in mitochondria from tissues where energy demand is high. Since the fine structure of cristae change with the metabolic states and energy demand, we hypothized that mitochondrial function was modulated by the molecules which controlled the morphology of cristae. Also the effect might relate to the genesis and development of age -related disorders. Immt (Inner Membrane Protein of Mitochondria, also called Mitofilin) locates on the inner membrane of mitochondria, characterized as a controller of the cristae shape in cells. To invest the physiological functions of Immt in mice, it is necessary to establish a line of Immt deficient mice.Gene-Trap is a gene targeting method with high efficiency. The bottleneck of gene targeting is the microinjection and screening for the chimeric mice which can breed heterozygote. Through the microinjection, we got the germline chimeric mice. We reconfirmed the heterozygote by the recombination of PCR and 5'RACE. The results from genomic PCR characterized the inserting position of gene-trap vector is in the third intron of the Immt gene. The heterozygote did not show any exterior differences. The offsprings of crossbreeding heterozygote contained one third Immt+/+ mice and two thirds heterozygote, and no Immt-/- was found after checking 346 offspring mice. Due to the Mendelian Principle in genetics, the ratio showed that the Immt-/- was not born.To examine the details of the lethal embryos, we check embryos at different development stages from intercrossing heterozygote. It showed that all the embryos survived through implantation but no homozygote was found at E10.5 stage. Using LacZ staing and HE staing methods, we found the Immt-/- embryos remained the E7.5 size and would not grow any more until they were absorbed after E10.5 while the heterozygote and Immt+/+ ones reached E9.5 size. The further structural analysis showed the Immt-/- could not form the cardiac structure and uncompletely closed neural tube. The TUNEL results showed that the Immt-/- exhibited more apoptosis than others at the same stage of E9.5 and reabsorbed.After examining the mitochondrial ultrastructure by electron microscope, we found the mitochondria in Immt-/- were enlarged with disorganized and partial vacuolated cristae. And enzyme histochemical staining confirmed severe mitochondrial dysfunction with decreased cytochrome c oxidase activity while the activity of succinate dehydrogenase had no detectable change. Measurements of respiratory chain enzyme activities in the liver of Immt+/- animals revealed significant reductions in the activities of COX while no obvious change in SDH. We also found that Immt-deficient mouse embryonic fibroblasts showed increased membrane potential (ΔΨm). What's more, the production of ROS in the Immt+/- MEF revealed increased. All these results confirmed severe mitochondrial OXPHOS dysfunction in Immt deficient mice.In summary, IMMT plays an important role in controlling the fine structure of critae and the loss of Immt causes the severe OXPHOS dysfunction and the embryonic lethality in mice. The Immt-/- embryos have smaller size, delayed neural development, and indistinct somites, also with absence of cardiac structure. All these data point to a role of IMMT in OXPHOS and emphasize the role of IMMT in life.