During the period covered by this report, we achieved several aims that were planned in the initial proposal.
One of the first goal of this proposal was to determine the molecular mechanism by which telomeres are attached to the nuclear envelope. In other words, which protein at telomeres and at the nuclear envelope trigger and/or maintain this attachment. For this, we conducted a mass spectrometry screen to identify in a unbiased manner the proteins that are connected to telomeres at this specific time of the cell cycle. We discovered that telomeres are more frequently found together with specific proteins from the nuclear envelope in late mitosis. We are now further analyzing these hits to determine their exact function in telomere tethering. In addition, we are using advanced super-resolution microscopy, the technology 3D-SIM, to get a better view on telomere organization at the nuclear envelope. Are telomeres tethered to specific structures such as nuclear pores (that connect the nucleoplasm and the cytoplasm of the cell), the nuclear lamina (a fine meshwork lining the inner side of the nuclear envelope, and giving mechanical support to the nucleus)? We found that telomeres colocalize with the reforming membrane, suggesting they could play an active role in post-mitotic nuclear assembly.
Two technological challenges that were very important for the progress of this work. First, we developed a technology that we called MadID, which probes telomere-nuclear envelope interaction in a semi-quantitative manner. And we achieved to add a fluorescent protein fused to a protein that specifically bind to telomeres, using CRISPR-Cas9 mediated genome editing.
Finally, our work highlighted a functional connection between telomeres, nuclear envelope integrity, and human aging.
