Visualization of Human Telomerase Localization by Fluorescence Microscopy Techniques

Human telomerase is a ribonucleoprotein (RNP) that synthesizes
DNA
repeats at the ends of chromosomes and maintains telomere length and genome stability. The enzyme comprises telomerase RNA (hTR) (which provides the template for telomere addition) and several protein subunits, including telomerase reverse transcriptase (hTERT) (the catalytic component). Intracellular trafficking of the enzyme has emerged as an important factor in the regulation of telomerase activity. Telomerase trafficking between nuclear Cajal bodies (proposed sites of telomerase biogenesis and regulation) and telomeres (sites of action) is regulated by the cell cycle in concordance with telomere synthesis during S phase. Here, we describe fluorescence microscopy approaches to visualize the subcellular localization of the essential RNA component of hTR relative to Cajal bodies and telomeres in cultured human cells. These methods include fluorescence in situ hybridization (to detect hTR and telomeric DNA) and immunofluorescence (IF) (to detect Cajal bodies and telomere-binding proteins). Because telomerase localization to telomeres is normally restricted to S phase, we also describe methods to synchronize and analyze cells within this phase of the cell cycle.