Ultrastructural immunolocalization of the senescence-related protein terminin in human fibroblasts

Terminin is a cytoplasmic protein originally found in human fibroblasts, where it can be present in three forms of different molecular weights: the 90 kDa form is synthesized in young fibroblasts, and is cleaved into a 60 kDa form in irreversibly growth-arrested senescent cells; a 30 kDa form of terminin is found in apoptotic cells. All the forms can be immunodetected by monoclonal antibody 1.2 in Western blots, whereas in immunohistochemistry the same antibody only recognizes the insoluble 60 and 30 kDa forms. Terminin immunopositivity may, therefore, be considered as a marker for cell senescence/ageing, terminal differentiation, and commitment to apoptosis, although the role(s) of this protein and its cleavage products in cell metabolism are still unknown. In the present investigation we aimed to elucidate the intracellular distribution of terminin in senescent human fibroblasts, using a combined immunohistochemical approach of light and transmission electron microscopy. For light microscopy, we used indirect immunodetection methods, utilizing either fluorochrome-labeled secondary antibodies or horseradish peroxidase (HRP)-conjugated antibodies finally revealed by incubation with diaminobenzidine (DAB); for transmission electron microscopy, we used either pre-embedding immunodetection with HRP-DAB or post-embedding gold-immunolabeling. All methods consistently identified cytoplasmic vacuoles as the main site of accumulation of immunodetectable terminin; based on their ultrastructural morphology, these membrane-bounded organelles are likely to be phagolysosomes or residual bodies.