OPA1 was originally identified as a regulator of mitochondrial IM fusion, so we decided to look at the mitochondrial network in our different cell lines. We found the mitochondrial network to be fragmented in the tetraCys OPA1 cells at similar levels as the OPA1 KO cells. We then set to analyse the mitochondrial ultrastructure. We performed TEM. Morphometric analyses showed that tetraCys OPA1 cells have an increased number of cristae junctions per mitochondrion, but reduced number of cristae per mitochondrion. Furthermore, we found reduced juxtaposition between the OM and the IM. The latter is important because it gave us the idea that the processing of OPA1 has a role in regulating the distance between both membranes, an important feature of the import process.
We then wondered whether the mitochondrial complexes that reside inside both the inner and the outer membrane were affected in these cells. We performed BN PAGE analysis in complexes extracted from isolated mitochondria. We found no defects on the assembly of neither the TOM40 complex proteins in our system. OPA1 locates in the IM, so we also tested the state of the main import complexes of proteins residing in either the IM, the IMS or the matrix. We found no differences in the assembly patterns of the TIM22 complex. On the other hand, the TIM23 complex imports proteins into the IMS, the IM and the matrix and is composed of the Tim23, Tim17 and Tim50 proteins. The BN PAGE showed a shifted pattern in the assembly of such a complex, which suggests that the composition of such a complex is different when the cells lack proteolytic processing of OPA1. Finally, in addition to the contact of the OM with the IM between the TOM40 and the TIM23 complexes, the MIB complex has been described. The MIB is primarily formed by contacts of the MICOS complex, in the IM, and the SAM complex in the OM. We found lower levels of the MIB complex in our tetraCys MEF, even after loading 50% more of the sample. These results suggest a relationship between the processing of OPA1 and the import of proteins into the mitochondria through the TIM23 complex. The TIM23 pathway requires both ATP and membrane potential to target and sort protein precursors into mitochondria. Among which are most of the respiratory chain subunits. In turn, proper function of the respiratory chain maintains optimal levels of both ATP and membrane potential. Thus, we looked at the assembly of the complexes and supercomplexes from the mitochondrial respiratory chain. We did not find apparent defects on complex II (SDH), complex III (UQCRC2), complex IV (Mt-CO1) or the ATPase (ATP5A). However, the assembly of supercomplexes appears to be affected as seen by the absence of the high molecular weight bands. Furthermore, a different band is detected in the tetraCys mitochondria on the ATP5A blot. We then looked at cell growth. Indeed, under normal growth conditions, i.e. using glucose as main C source, no difference was observed. On the other hand, when growth under respiratory conditions, i.e. galactose as the main C source, both tetraCys OPA1 and OPA1 KO MEF did not grow. These results suggest problems in the mitochondrial respiration or handling oxidative stress, present under respiratory conditions. Our analysis of the mitochondrial complexes also indicated that the assembly of TIM23 complex is different when no OPA1 processing happens. We then perform import experiments into the matrix in whole cells. We took advantage of the host lab’s expertise and used super resolution microscopy and the localisation index concept described in Petronilli, V. et. al. 2001. We observed an almost complete overlap on the WT OPA1 MEF, but a spread signal of mito-YFP in both the tetraCys OPA1 and the OPA1 KO MEF, which means that the import of the mito-YFP is impaired. As mentioned before, TIM23-import depends on the mitochondrial membrane potential. When we treat the OPA1 WT cells with FCCP, a mitochondrial uncoupler, we observed impaired import of mito-YFP, thus probing the efficacy of the assay to show mitochondrial import.
