Changes in lamina structure are followed by spatial reorganization of heterochromatic regions in caspase-8-activated human mesenchymal stem cells

Vered Raz,Rob C Hoeben,Bart J Vermolen,Yuval Garini,Willem C R Sloos,Ian T Young,Shoshan KnaäN-Shanzer,FrançOise Carlotti,Roeland W Dirks,Egge Van Der Poel,Hans J Tanke,Antoine A F De Vries

doi:10.1242/jcs.03180

Abstract

Apoptosis is fundamental to the regulation of homeostasis of stem cells in vivo. Whereas the pathways underlying the molecular and biochemical details of nuclear breakdown that accompanies apoptosis have been elucidated, the precise nature of nuclear reorganization that precedes the demolition phase is not fully understood. Here, we expressed an inducible caspase-8 in human mesenchymal stem cells, and quantitatively followed the early changes in nuclear organization during apoptosis. We found that caspase-8 induces alteration of the nuclear lamina and a subsequent spatial reorganization of both centromeres, which are shifted towards a peripheral localization, and telomeres, which form aggregates. This nuclear reorganization correlates with caspase-3 sensitivity of lamina proteins, because the expression of lamin mutant constructs with caspase-3 hypersensitivity resulted in a caspase-8-independent appearance of lamina intranuclear structures and telomere aggregates, whereas application of a caspase inhibitor restrains these changes in nuclear reorganization. Notably, upon activation of apoptosis, we observed no initial changes in the spatial organization of the promyelocytic leukemia nuclear bodies (PML-NBs). We suggest that during activation of the caspase-8 pathway changes in the lamina structure precede changes in heterochromatin spatial organization, and the subsequent breakdown of lamina and PML-NB.

Highlights

Maintenance of the pool of stem cells is under tight control in vivo and is balanced by the rate of cell proliferation, differentiation and apoptosis (Lawen, 2003)
Cleavage of the lamina proteins is crucial for cell death because the structural support of the nucleus depends on an intact lamina structure
We confirm the findings of previous studies (Broers et al, 2002; Ruchaud et al, 2002), showing that lamina reorganization during activation of apoptosis precedes chromatin breakdown, and we extend these observations by showing that it depends on caspase-3 activation

Summary

Introduction

Maintenance of the pool of stem cells is under tight control in vivo and is balanced by the rate of cell proliferation, differentiation and apoptosis (Lawen, 2003). The activated effector caspases cleave cellular substrates, leading to membrane blebbing, DNA degradation and nucleus fragmentation (Wyllie et al, 1981; Nagata, 2005). Proteins of the nuclear envelope, which support the nuclear architecture, are directly cleaved by the effector caspases (Lazebnik et al, 1995). Cleavage of the lamina proteins is crucial for cell death because the structural support of the nucleus depends on an intact lamina structure. During the nuclear breakdown phase, cleavage of the lamina proteins precedes DNA condensation, which is subsequently fragmented (Rao et al, 1996). It was suggested that chromatin condensation during apoptosis depends on degradation of lamin A (Ruchaud et al, 2002). The changes in nuclear organization that precede the nuclear breakdown phase were so far poorly studied

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