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MULTILEVEL ANALYSIS OF NUCLEAR DYNAMICS IN LAMIN PERTURBED HUMAN FIBROBLASTS

Abstract

The nuclear lamina provides structural support to the nucleus and has a central role in defining nuclear organization. Defects in its filamentous constituents, the lamins, lead to a class of diseases collectively referred to as laminopathies. On the cellular level, lamin mutations affect the physical integrity of nuclei and nucleo-cytoskeletal interactions, resulting in increased susceptibility to mechanical stress and altered gene expression [1]. Most studies regarding the mechanical properties of the nucleus in laminopathic conditions are based on the induction of extracellular stress, such as strain or compression, and focus on nuclear integrity and/or nucleo-cytoskeletal interaction [2]. Far less is known about the role of nuclear organization and mobility under basal steady-state conditions.

In this study, we quantitatively compared nuclear organization, nuclear deformation and chromatin mobility of fibroblasts from a Hutchinson-Gilford progeria patient with cells from a lamin A/C-deficient patient and wild-type dermal fibroblasts. To this end, we created a toolbox in imageJ for automatically analyzing both nuclear as well as subnuclear dynamics in living cells. Simultaneously, we developed a workflow for comparing cellular morphology and subcellular protein distribution in a high content fashion.

We found that the absence of functional lamin A/C leads to increased nuclear plasticity on the hour and minute time scale but also to increased intranuclear mobility down to the seconds time scale. In contrast, progeria cells showed overall reduced nuclear dynamics. In addition, high content analysis revealed marked morphological and topological differences between different culture passages within a cell type and between different pathological variants of culture-age matched laminopathic cell types.

(1) Broers J et al. (2006). Physiol Rev. 86:967-1008. (2) Dahl K. et al. (2008). Circ.Res. 102:1307-1318.

(3) Hoebe R et al. (2007). Nat Biotech 25 (2007), p. 249. (4) De Vos W et al. (2010). BBA 1800(4), p.48.

Keywords

nuclear shape, nuclear mobility, tracking, high content analysis

Administrative data

Presenting author: Winnok H. De Vos
E-mail: winnok.devos@ugent.be
Organisation: University of Ghent (Belgium)

co-authors: F. Houben (1), J. Broers (1), R.A. Hoebe (2), E.M.M. Manders (2), F. Ramaekers (1) and P. Van Oostveldt (3)

(1). University of Maastricht (The Netherlands)

(2). University of Amsterdam (The Netherlands)

(3). University of Ghent (Belgium)

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