Linking Cell Differentiation, Death, and Mitochondrial Function
We are only now beginning to understand the complex regulatory processes that control the differentiation of pluripotent stem cells (PSCs), and we know even less about how the differentiation process alters the structure and function of cellular organelles. Of particular interest in this regard are the mitochondria, which are present in a fragmented state in PSCs but fuse into more elongated forms in differentiated cells. Differentiation is also accompanied by an increased resistance to apoptotic cell death, which is regulated at the level of the mitochondria by the BCL-2 family of proteins. This apparent connection led Vanderbilt Basic Sciences investigator Vivian Gama and her laboratory to hypothesize that in addition to apoptosis, BCL-2 family proteins modulate mitochondrial dynamics, the processes of fission and fusion that determine the overall degree of mitochondrial fragmentation. To test this hypothesis, the researchers began with the discovery that differentiation of human embryonic stem cells (hESCs) is accompanied by a decrease in expression of the BCL-2 family member MCL-1. This was an unexpected finding because MCL-1 is an anti-apoptotic protein, yet as its expression dropped, the cells became more rather than less resistant to apoptosis. Then, using several small molecule inhibitors of various BCL-2 family members, the investigators showed that MCL-1 blockade led to a reduction in expression of OCT4 (a marker of pluripotency), decreased sensitivity to DNA damage-induced cell death, and increased mitochondrial fusion. They obtained similar findings when they blocked MCL-1 expression in hESCs using siRNA. Reduced MCL-1 activity also led to a reduction in phosphorylation (and activation) of the protein DRP-1, which is primarily responsible for mitochondrial fission. Co-immunoprecipitation and proximity ligation studies demonstrated that MCL-1 interacts directly with DRP-1 at the outer mitochondrial membrane, thereby suggesting that MCL-1 promotes fission by facilitating DRP-1 phosphorylation. Somewhat unexpectedly, the researchers used the same methods to show that MCL-1 in the mitochondrial matrix also interacts with OAT1, which is primarily responsible for mitochondrial fusion. In fact, decreased MCL-1 expression during differentiation was accompanied by decreased OAT1 levels, despite the fact that mitochondrial fusion increased under those conditions. Despite this apparent discrepancy, the researchers showed that selective expression of MCL-1 at the outer mitochondrial membrane regulates apoptosis and promotes mitochondrial fission, whereas expression of MCL-1 in the mitochondrial matrix stabilizes OAT1 and suppresses differentiation. The findings demonstrate a previously unappreciated function of MCL-1, regulation of mitochondrial dynamics, that is distinct from its role in modulating apoptosis. They also define a link between differentiation and mitochondrial dynamics that will be the subject of interesting further exploration. The work is published in the journal Stem Cell Reports [Rasmussen et al. (2018), Stem Cell Rep. https://doi.org/10.1016/j.stemcr.2018.01.005]