Live-cell imaging of telomere repair reveals how telomerase diffuses and repeatedly engages in transient interactions along telomere lengths before stably engaging telomere ends. [University of Colorado Cancer Center]
Live-cell imaging of telomere repair reveals how telomerase diffuses and repeatedly engages in transient interactions along telomere lengths before stably engaging telomere ends. [University of Colorado Cancer Center]

A pair of techniques—CRISPR gene editing and single-molecule microscopy—have been put together to subject telomere repair to a new level of scrutiny, one that not only zooms in on single proteins, but also tracks the proteins over time. The proteins, fluorescently labeled versions of telomerase, were observed displaying a kind of probing behavior, rapidly sticking and unsticking from telomeres, before ultimately sticking more firmly to telomere ends, and only then adding a repeating DNA sequence to the repeating DNA sequences that make up telomeres.

These observations reveal that a kind of two-step diffusion process takes place during telomere repair. In the first step, telomerase diffuses evenly through the nucleus. When it happens to hit anywhere on a telomere, it sticks briefly, thus increasing the concentration of telomerase near telomeres. In the second step, telomerase—already in the neighborhood of the telomere ends—diffuses again, but this time with an increased chance of hitting a telomere end, where a secure and relatively long-lived attachment can be made.

These telomere repair dynamics were uncovered by scientists based at the University of Colorado. They used CRISPR DNA-editing technology to insert code into the gene that makes telomerase. This inserted code manufactured a fluorescent protein, which was attached to telomerase. The scientists then used what some call nanoscopy to see this fluorescent protein.

Details of the work appeared August 11 in the journal Cell, in an article entitled, “Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres.” The article made a point of noting that in cancer research, telomere repair is of particular interest, because it can endow cancer cells with immortality. A better understanding of telomere repair dynamics, the article’s authors realized, could point to more effective therapies against cancer.

“We demonstrate that telomerase uses three-dimensional diffusion to search for telomeres, probing each telomere thousands of times each S-phase but only rarely forming a stable association,” the article’s authors wrote. “Both the transient and stable association events depend on the direct interaction of the telomerase protein TERT [telomerase reverse transcriptase] with the telomeric protein TPP1.”

Without live-cell imaging, a version of which was used in the current study, looking close enough to see the fluorescence of a single protein would have required “fixing” the cell and visualizing it with a microscope. It would have been a snapshot. The ability to see the processes inside a live cell at this magnification is like shooting video.

“The amazing thing to me is that 3 years ago, you couldn't have done any of this. This is how fast things are moving in biology, just rocketing ahead,” said the article’s senior author, Thomas Cech, Ph.D., CU Boulder Distinguished Professor, Nobel laureate, and director of CU's BioFrontiers Institute. “With this fixed cell imaging, we didn't see the dynamics. And the rapidly diffusing telomerase is invisible, washed out, or lost in the background.”

Dr. Cech suggests that this technique of CRISPR-aided nanoscopy will likely be used by scientists outside the field of telomere research. He also hopes this specific finding will aid in screening anti-telomerase drugs.

“Right now we don't have a great telomerase inhibitor. We don't know at which step our first generation of these drugs is interfering so we don't know how to optimize these drug candidates for anticancer effect,” Dr. Cech explained. “Does a drug prevent the assembly of telomerase? Does it keep telomerase from moving near telomeres? Does it prevent probing? Does it prevent telomerase from finding a telomere end?

“Knowing where a drug blocks the ability of telomerase to lengthen telomeres could have broad applicability for diverse cancers,” Dr. Cech added.

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