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. 2012 Feb;4(2):119-32.
doi: 10.18632/aging.100434.

Automated image analysis of nuclear shape: what can we learn from a prematurely aged cell?

Meghan K Driscoll  1 , Jason L Albanese, Zheng-Mei Xiong, Mitch Mailman, Wolfgang Losert, Kan Cao
Affiliations

Automated image analysis of nuclear shape: what can we learn from a prematurely aged cell?

Meghan K Driscoll et al. Aging (Albany NY). 2012 Feb.

Abstract

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.

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Conflict of interest statement

The authors of this manuscript have no conflict of interest to declare.

Figures

Figure 1
Figure 1. The boundary curvature of HGPS and normal nuclei
(a) The curvature of nuclei is automatically extracted from fluorescence images of anti-laminA/C immunostaining. Here, the curvature of HGPS and normal nuclei is shown as a colored outline, where blue represents regions of large positive curvature, and red regions of large negative curvature (scale bar: 10 μm). Blebbed nuclei have more regions of negative curvature, and so have more red signals. (b) High magnification examples of the extracted boundary curvature of a blebbed, HGPS nucleus, and a more oval, normal nucleus (scale bar: 10 μm). (c) The boundary curvature of hundreds of nuclei can be represented in a single heat map. In these heat maps, which here correspond to two HGPS cell lines (HGPS1 and HGPS2, respectively) and one control cell line (Normal), each vertical line is the stretched, colored outline of a single nucleus. Regions of large negative curvature are colored blue while regions of large negative curvature are colored red. (d) The nuclei are ordered from left to right by increasing mean negative curvature (MNC), which is shown in the line plots. (e) The MNC of populations from both HGPS cell lines is statistically different from the population from the normal control, as illustrated in this histogram.
Figure 2
Figure 2. The nuclear morphology and progerin levels of rapamycin and RAD001 treated HGPS cells
(a) The phenotype of nuclear blebbing was improved in RAD001 and rapamycin treated HGPS fibroblast cells. Cells were stained with DAPI (blue), laminA/C antibody (red), and progerin antibody (green) to show nuclear location and morphology. The treatment duration is for seven weeks. Mock: vehicle (DMSO, 0.025% v/v); Rap: 0.68 μM rapamycin, Rad: 0.1 μM RAD001. (Scale bar: 10 μm) (b) Quantification of the percentage of blebbing in all treatments. At least 200 nuclei were counted blindly. M: vehicle (DMSO, 0.025% v/v); Rap: 0.68 μM rapamycin; RAD100: 0.1 μM RAD001 treatment; RAD500: 0.5 μM RAD001 treatment. (c) Progerin was decreased in rapamycin or RAD001 treated HGPS fibroblasts. The relative amount of progerin was quantified using quantitative western blotting analysis and compared to the mock-treated HGPS cells.
Figure 3
Figure 3. Imaging analysis of RAD001 and rapamycin treated HGPS cells
(a) Heat maps that represent the boundary curvature of the HGPS cells treated with the vehicle (mock), 100 nM and 500 nM RAD001 (Rad), and rapamycin (Rap). (b) MNC of populations from the mock treated HGPS cell lines is statistically different from the populations of RAD001 or rapamycin treated cells (p < 0.001) (c-e) Compared to the mock treated nuclei, the RAD001 and rapamycin treated nuclei have smaller area (c) and fewer invaginations (d), but similar eccentricity (e).
Figure 4
Figure 4. RAD001 induces a gradual change in nuclear morphology in a dosage dependant manner
(a) Heat maps that represent the boundary curvature of HGPS cells treated with the vehicle (mock) or 20 or 60 nM RAD001. (b) Both doses significantly reduce the mean negative curvature as shown by the histogram. (c) The area of mock treated nuclei is greater than both doses of RAD001 treated nuclei, but the nuclei that received the smaller dose of RAD001 have greater area than the nuclei that received the larger dose. The same trend in area change is apparent to the same extent in the treated control normal fibroblasts.
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