Telomerase reverse transcriptase in motion - dynamics of subcellular localization and functional consequences in aging and senescence

端粒酶通过抵消端粒缩短在衰老过程中发挥核心作用。其催化亚基端粒酶逆转录酶（TERT）除了保存端粒的任务外，还具有非端粒功能。已经证明，TERT存在于线粒体中，在那里它具有保护作用。值得注意的是，心血管系统的有丝分裂后细胞在两个隔室中都具有TERT。在某些条件下，它从细胞核中输出，伴随着线粒体中的增加。到目前为止，还不清楚离开细胞核的相同的TERT分子是否进入线粒体。结合该提案中合作伙伴的独特专业知识和方法，将首次允许在单细胞中跟踪现有的TERT分子，也是在与细胞衰老，衰老和年龄相关疾病相关的条件下。该项目的一个目的是跟踪已经存在的与可光转换的Dendra 2蛋白融合的TERT分子，该蛋白在照射后改变其荧光特性，从而可以在活细胞中与新合成的分子区分开来。这将在稳定表达TERT-Dendra 2的不同增殖能力的几种细胞类型中进行，并且另外通过显微注射重组蛋白。为了研究TERT以非经典方式从细胞核转移到线粒体的遥远可能性，我们将破坏TERT中的线粒体靶向序列以防止线粒体通过线粒体膜的易位酶输入。上述线粒体TERT的增加导致该蛋白质在不同水平上影响线粒体内过程的假设。因此，我们将通过Förster共振能量转移，单分子定位显微镜和免疫沉淀来研究TERT是否与线粒体转录因子A和锰超氧化物歧化酶相互作用，因为所有三种蛋白质都已被证明与线粒体DNA相关。此外，将通过对来自完全缺乏TERT、在细胞核和线粒体中含有TERT或仅在线粒体中含有TERT的细胞的线粒体RNA进行全面分析来探索TERT对线粒体转录组的影响。此外，呼吸链的变化将通过O2消耗和呼吸链超复合体组装水平进行检查。最后，我们将用与细胞衰老、衰老和年龄相关疾病相关的刺激物治疗心肌细胞和心脏成纤维细胞，并通过生命细胞成像跟踪TERT的旅行;这些跟踪实验将在生物化学上得到反映。在功能方面，我们将测量呼吸、凋亡、肥大、衰老和肌成纤维细胞分化潜力。这些研究将首次揭示现有TERT分子的动态分布及其在衰老，衰老和年龄相关疾病中的细胞后果。

Telomerase plays a central role in aging processes by counteracting telomere shortening. Its catalytic subunit Telomerase Reverse Transcriptase (TERT) has, besides its task in telomere-preservation, also non-telomeric functions. It has been demonstrated that TERT is present in mitochondria, where it has a protective role. Of note, post-mitotic cells of the cardiovascular system possess TERT in both compartments. Under certain conditions, it is exported from the nucleus with a concomitant increase in the mitochondria. Up to now it is unclear, if the same TERT molecules that leave the nucleus enter the mitochondria. Combining the unique expertise and methods of the partners in this proposal will allow for the first time to follow existing TERT molecules in single cells, also under conditions relevant for cellular senescence, aging and age-related diseases. One aim of this project is to track already existing TERT molecules fused to the photoconvertible Dendra2 protein, which after irradiation changes its fluorescence properties and can thereby be distinguished from newly synthesized molecules, in living cells. This will be done in several cell types of different proliferation capacity stably expressing TERT-Dendra2 and, additionally, by microinjection of the recombinant protein. To study the remote possibility of a transfer of TERT from the nucleus to the mitochondria in a non-canonical way, we will destroy the mitochondrial targeting sequence in TERT to prevent mitochondrial import via the translocases of the mitochondrial membranes. The above-described increase in mitochondrial TERT leads to the hypothesis that this protein affects intra-mitochondrial processes at various levels. Therefore, we will investigate by Förster Resonance Energy Transfer, Single Molecule Localization Microscopy and immunoprecipitation, whether TERT interacts with Mitochondrial Transcription Factor A and Manganese Superoxide Dismutase, as all three proteins have been demonstrated to be associated with mitochondrial DNA. Furthermore, the impact of TERT on the mitochondrial transcriptome will be explored by comprehensive profiling of mitochondrial RNAs from cells completely devoid of TERT, containing TERT in the nucleus and the mitochondria or exclusively in the latter. In addition, changes in the respiratory chain will be examined by O2-consumption and at the level of respiratory chain supercomplex assembly. Finally, we will treat cardiomyocytes and cardiac fibroblasts with stimuli relevant for cellular senescence, aging and age-related diseases and follow TERT travel by life cell imaging; these tracking experiments will be mirrored biochemically. On the functional side, we will measure respiration, apoptosis, hypertrophy, senescence and myofibroblast differentiation potential. These studies will for the first time shed light on the dynamic distribution of existing TERT molecules and its cellular consequences in senescence, aging and age-related diseases.