Telomerase reverse transcriptase (TERT) is a protein that catalyzes the reverse transcription of telomere elongation. TERT is also expected to play a noncanonical role beyond telomere lengthening since it localizes not only in the nucleus but also in mitochondria, where telomeres do not exist. Several studies have reported that mitochondrial TERT regulates apoptosis induced by oxidative stress. However, there remains controversy about whether mitochondrial TERT promotes or inhibits apoptosis, mainly due to the lack of information on changes in the TERT distribution in individual cells over time. Here we simultaneously detected apoptosis and TERT localization after oxidative stress in individual HeLa cells by live-cell tracking. This tracking revealed that the stress-induced accumulation of TERT in mitochondria resulted in apoptosis but that the accumulation positively correlated with the time until cell death. The results suggest a new model in which mitochondrial TERT has two opposing effects at different stages of apoptosis: it predetermines apoptosis at the first stage of cell-fate determination but also delays apoptosis at the second stage. Because these distinct effects respectively support both sides of the controversy regarding the role of mitochondrial TERT in apoptosis, our model integrates two opposing hypotheses. Furthermore, detailed statistical analysis of TERT mutations, which have been predicted to inhibit TERT transport to mitochondria, revealed that these mutations suppress apoptosis independent of the mitochondrial localization of TERT. Together, these results indicate that the non-canonical functions of TERT affect a wide range of apoptotic pathways.

Cancer cells exhibit the unique characteristics of high proliferation and aberrant DNA damage response, which prevents cancer therapy from effectively eliminating them. The machinery required for telomere maintenance, such as telomerase and the alternative lengthening of telomeres (ALT), enables cancer cells to proliferate indefinitely. In addition, the molecules in this system are involved in noncanonical pro-tumorigenic functions. Of these, the function of the cyclic GMP–AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, which contains telomere-related molecules, is a well-known contributor to the tumor microenvironment (TME). This review summarizes the current knowledge of the role of telomerase and ALT in cancer regulation, with emphasis on their noncanonical roles beyond telomere maintenance. The components of the cGAS-STING pathway are summarized with respect to intercell communication in the TME. Elucidating the underlying functional connection between telomere-related molecules and TME regulation is important for the development of cancer therapeutics that target cancer-specific pathways in different contexts. Finally, strategies for designing new cancer therapies that target cancer cells and the TME are discussed.

SHORT ABSTRACTThis paper summarizes how to visualize the flexible inter-domain movements of CRISPR-associated protein Cas9 using single molecule FRETLONG ABSTRACTThe CRISPR-associated protein Cas9 is widely used as a genome editing tool because of its ability to be programmed to cleave any DNA sequence that is followed by a protospacer adjacent motif. The continuing expansion of Cas9 technologies has stimulated studies regarding the molecular basis of the Cas9 catalytic process. Here we summarize methods for single molecule FRET (smFRET) to visualize the inter-domain movements of Cas9 protein. Our measurements and analysis demonstrate flexible and reversible movements of the Cas9 domains. Such flexible movements allow Cas9 to adopt transient conformations beyond those solved by crystal structures and play important roles in the Cas9 catalytic process. In addition to the smFRET measurement itself, to obtain precise results, it is necessary to validate Cas9 catalytic activity. Also, fluorescence anisotropy data are required to interpret smFRET data properly. Thus, in this paper, we describe the details of these important additional experiments for smFRET measurements.

Telomerase reverse transcriptase (TERT) is a protein that catalyzes the reverse transcription of telomere elongation. TERT is also expected to play a non‐canonical role beyond telomere lengthening since it localizes not only in the nucleus but also in mitochondria, where telomeres do not exist. Several studies have reported that mitochondrial TERT regulates apoptosis induced by oxidative stress. However, there is still some controversy as to whether mitochondrial TERT promotes or inhibits apoptosis, mainly due to the lack of information on changes in TERT distribution in individual cells over time. Here, we simultaneously detected apoptosis and TERT localization after oxidative stress in individual HeLa cells by live‐cell tracking. Single‐cell tracking revealed that the stress‐induced accumulation of TERT in mitochondria caused apoptosis, but that accumulation increased over time until cell death. The results suggest a new model in which mitochondrial TERT has two opposing effects at different stages of apoptosis: it predetermines apoptosis at the first stage of cell‐fate determination, but also delays apoptosis at the second stage. As such, our data support a model that integrates the two opposing hypotheses on mitochondrial TERT's effect on apoptosis. Furthermore, detailed statistical analysis of TERT mutations, which have been predicted to inhibit TERT transport to mitochondria, revealed that these mutations suppress apoptosis independent of mitochondrial localization of TERT. Together, these results imply that the non‐canonical functions of TERT affect a wide range of mitochondria‐dependent and mitochondria‐independent apoptosis pathways.