Date Published: February 16, 2012
Publisher: Impact Journals LLC
Author(s): Meghan K. Driscoll, Jason L. Albanese, Zheng-Mei Xiong, Mitch Mailman, Wolfgang Losert, Kan Cao.
The premature aging disorder, Hutchinson-Gilford progeria syndrome (HGPS), is caused by mutant lamin A, which affects the nuclear scaffolding. The phenotypic hallmark of HGPS is nuclear blebbing. Interestingly, similar nuclear blebbing has also been observed in aged cells from healthy individuals. Recent work has shown that treatment with rapamycin, an inhibitor of the mTOR pathway, reduced nuclear blebbing in HGPS fibroblasts. However, the extent of blebbing varies considerably within each cell population, which makes manual blind counting challenging and subjective. Here, we show a novel, automated and high throughput nuclear shape analysis that quantitatively measures curvature, area, perimeter, eccentricity and additional metrics of nuclear morphology for large populations of cells. We examined HGPS fibroblast cells treated with rapamycin and RAD001 (an analog to rapamycin). Our analysis shows that treatment with RAD001 and rapamycin reduces nuclear blebbing, consistent with blind counting controls. In addition, we find that rapamycin treatment reduces the area of the nucleus, but leaves the eccentricity unchanged. Our nuclear shape analysis provides an unbiased, multidimensional “fingerprint” for a population of cells, which can be used to quantify treatment efficacy and analyze cellular aging.
Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease that occurs in approximately 1 out of 4 million live births . Visible symptoms of patients with HGPS include a pronounced forehead, short stature, receding mandible, conspicuous veins in the scalp, alopecia and diminished subcutaneous fat [1-4]. Internally, such patients undergo accelerated organ degeneration. The average life expectancy of HGPS patients is just 14 years, with death typically resulting from heart attacks or stroke [1-4].
One of the hallmarks of HGPS is the abnormal nuclear shape known as blebbing. This has been the main morphological feature identifying an HGPS cell line and has been used to determine the effectiveness of treatments for HGPS. The traditional method of measuring blebbing is a manual, blind count of the percentage of blebbed nuclei. However, this method has no standard criteria and is extremely time consuming. Sorting the nuclei into two categories, normal and blebbed, also obscures the fact that blebbing is not an either/or phenomenon, but varies continuously. The subjectivity and variability of the threshold for blebbed nuclei makes it impossible to compare values obtained by different counters. The need for an unbiased, quantitative method of measuring the degree of blebbing in a cell sample is clear.