David W. Knowles, Sophie A. Lelièvre+,
Carlos Ortiz de Solórzano,
Stephen J. Lockett++, Mina J. Bissell, Damir Sudar
Life Sciences Division, Lawrence Berkeley National
Laboratory, Berkeley, California 94720
+Department of Basic Medical Sciences, Purdue University, West Lafayette, IN 47907-1246
++SAIC-Frederick, MD 21702
The extracellular matrix (ECM) plays a critical role in directing cell behaviour and morphogenesis by regulating gene expression and nuclear organization. Using non-malignant (S1) human mammary epithelial cells (HMECs), it was previously shown that ECM-induced morphogenesis is accompanied by the redistribution of nuclear mitotic apparatus (NuMA) protein from a diffuse pattern in proliferating cells, to a peripherial multi-focal pattern as HMECs growth arrested and completed morphogenesis . A process taking 10 to 14 days.
To further investigate the link between NuMA distribution and the growth stage of HMECs, we have studied the distribution of NuMA in non-malignant S1 cells and their malignant, T4, counter-part using a novel model-based image analysis technique. This technique, based on a multi-scale Gaussian blur analysis (Figure 1), quantifies the size of punctate features in an image. Cells were cultured in the presence and absence of a reconstituted basement membrane (rBM) and imaged in 3D using confocal microscopy, for fluorescently labeled monoclonal antibodies to NuMA (faNuMA) and fluorescently labeled total DNA. Nuclear segmentation , based on the DNA staining, allowed NuMA distribution analysis to be contained within the nuclei of individual cells.
faNuMA distribution was clearly less punctate in non-malignant S1 cells in rBM culture at day 4 (proliferation) than at day 10, when these cells complete differentiation, as previously reported. Interestingly, the distribution of faNuMA in malignant T4 cells, in rBM culture at day 4, was measurably less punctate than that in S1 cells at either time. This supports the phenotypic trait of T4 cells cultured in rBM which is their inability to differentiate. In the absence of rBM, non-malignant S1 cells are also unable to differentiate but proliferate in presence and growth arrest in absence of epithelial growth factor (EGF). Again, there was a clear measurable difference between faNuMA distribution which was less punctate during proliferation and more punctate when these cells growth arrested. Interestingly, in the absence of rBM there was no measurable difference in faNuMA distribution in proliferating S1 cells and their malignant counterpart which also possibly reflects the inability of either cell type to differentiate.
The ability to discern cell phenotype based on quantifyable molecular analysis, in this case the spatial distribution of a nuclear protein, has broad application in furthering fundamental understanding of biological processes.
 Sophie A. Lelièvre, et al 1998
Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus
PNAS 1998 95: 14711-14716.
 Ortiz de Solórzano C.,et al 1999
Segmentation of Confocal Microscope Images of Cell Nuclei in Thick Tissue Sections
Journal of Microscopy, 193(3):212-226