Non-apoptotic mitochondrial functions of C. elegans CED-9 BCL2
Final Report Abstract
The anti-apoptotic BCL2-like protein CED-9 of C. elegans localizes to the outer mitochondrial membrane (OMM) and prevents apoptosis in cells ‘fated’ to live. We had demonstrated that the expression of ced-9 in C. elegans embryos alters steady state mitochondrial morphology and that loss-of-function or gain-of-function mutations of ced-9 also affect mitochondrial morphology. Based on these observations, we had proposed that CED-9 BCL2 has non-apoptotic mitochondrial function(s) and this formed the basis of this application. To investigate these non-apoptotic mitochondrial function(s), we proposed to measure – in ced-9 mutants - rates of mitochondrial fusion and fission (Objective 1), to analyse various aspects of mitochondria function (Objective 2) and to determine the composition and function of CED-9-containing complexes on the OMM (Objective 3). As outlined in more detail below, as a result of very low rates of mitochondrial fusion and fission in C. elegans embryos as well as technical difficulties, we were unable to determine the role of ced-9 BCL2 in mitochondrial dynamics. However, in the process, we discovered that mitochondrial morphology in body wall muscle cells dramatically changes with increasing age and this formed the basis of a subsequent study that was published in 2015. We ran into difficulties of a different nature in Objective 2 and were unable to obtain conclusive and reproducible data on the role of ced-9 BCL2 in mitochondrial function. This objective eventually had to be abandoned. Nevertheless, tools developed for this objective were critical in our analyses of the mitochondrial unfolded protein response, which has so far resulted in two publications. Finally, as a result of CED-9 BCL2 protein most likely being part of a multi-protein complex on the OMM and inaccessible to antibodies, we also ran into technical difficulties in our effort to pull-down CED-9 BCL2-containing complexes from C. elegans lysates. However, we eventually identified 17 proteins that were pulled-down specifically with CED-9 but not with controls. Some of the proteins identified, we are currently following up on, and I am optimistic that this will reveal additional novel functions of CED-9 BCL2 in mitochondrial biology. Specifically, we are in the process of analysing mitochondrial inheritance in asymmetrically dividing cells and are investigating the role of CED-9 BCL2 and its interactors in this process (now funded by the BBSRC). Finally, our aim also was to characterize in more detail CED-9 BCL2-containing complexes through super resolution imaging. This also turned out to be more challenging than anticipated, especially with respect to existing technology. However, at our current institution, we have developed methodology to perform live cell imaging close to super resolution, and we will use this to investigate mitochondrial dynamics and function as well as CED-9 BCL2 in more detail in the future.
Publications
- 2014. Age-dependent changes in mitochondrial morphology and volume are not predictors of lifespan. Aging (Albany NY). 2014 Feb;6(2):118-30
Regmi SG, Rolland SG, Conradt B
(See online at https://doi.org/10.18632/aging.100639) - 2015. The loss of LRPPRC function induces the mitochondrial unfolded protein response. Aging (Albany NY), Vol. 7. 2015, Issue 9, pp. 701-712.
Köhler, F., Müller-Rischart, A.K., Conradt, B., Rolland, S.G.
(See online at https://doi.org/10.18632/aging.100812) - 2019. Compromised Mitochondrial Protein Import Acts as a Signal for UPRmt. Cell Reports, Vol. 28. 2019, Issue 7, pp. 1659-1669.e5.
Rolland SG, Schneid S, Schwarz M, Rackles E, Fischer C, Haeussler S, Regmi SG, Yeroslaviz A, Habermann B, Mokranjac D, Lambie E, Conradt B
(See online at https://doi.org/10.1016/j.celrep.2019.07.049) - 2020. Autophagy compensates for defects in mitochondrial dynamics. PLoS Genet. Vol. 16. 2020, Issue 3: e1008638.
Haeussler S., Köhler F., Witting M., Premm M.F., Rolland S.G., Fischer C., Chauve L., Casanueva O., Conradt B.
(See online at https://doi.org/10.1371/journal.pgen.1008638)
