Research Article: Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay

Date Published: October 16, 2011

Publisher: Impact Journals LLC

Author(s): Marco De Cecco, Jessie Jeyapalan, Xiaoai Zhao, Mimi Tamamori-Adachi, John M. Sedivy.



Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.

Partial Text

Normal somatic cells, with the exception of germ cells and some stem cells, display a finite replicative capacity. This phenomenon was first described in cultured human fibroblasts [1], and is commonly referred to as replicative cellular senescence. Subsequently, these observations were extended to a wide variety of vertebrate species and cell types, and senescence is now believed to be a general property of replicative cells [2]. Cellular senescence has been studied predominantly in cell culture (in vitro), although evidence is accumulating that this process also occurs in intact organisms (in vivo) under a variety of pathological and normal conditions [3-5]. A well studied trigger of cellular senescence is the shortening of telomeres [6], but many other stimuli, most importantly the activation of oncogenes and a variety of genotoxic stresses, have also been documented [7]. The phenotypes of senescent cells affect most, if not all aspects of cellular physiology, including gene expression, chromatin organization, protein processing and metabolism [3, 8]. Given the extensive, complex, and often cell-type specific nature of these changes this remains an active area of investigation.

Morphological changes were among the first reported phenotypes of senescent cells [1]. Changes in cell shape, volume and macromolecular content (DNA, RNA, protein) were actively studied in the 1970’s but have received little attention since. Unfortunately, some of the original literature is not readily accessible, such as the increase in total nuclear protein of senescent HDF, which is noted in the CRC Handbook of Cell Biology of Aging [34], but the original reference [29] is not listed on PubMed. Although the increased protein content of senescent cells in vitro is in general well accepted, whether this occurs in vivo is not known. Our objective was to investigate changes in total protein as a potential new biomarker of aging, but this necessitated first the development of a fluorescence microscopy method to detect and quantify any changes.