Active relocation of chromatin and endoplasmic reticulum into blebs in late apoptotic cells

Abstract

Plasma membrane blebbing is a defining characteristic of apoptosis, but its significance is not understood. Using live-cell imaging we have identified two phases of apoptotic blebbing. The early phase is restricted to adherent cells, and is prevented by the Rho-activated kinase inhibitor Y27632. The late phase is partially resistant to Y27632, and generates morphologically distinct membrane protrusions that are likely precursors to apoptotic bodies. Late blebbing is observed in all apoptotic cells tested. It occurs at a fixed period before phosphatidyl serine exposure, indicating that it is a universal and important feature of apoptosis. Late blebs contain a cortical layer of endoplasmic reticulum that often surrounds condensed chromatin, while other organelles are excluded. The appearance in some apoptotic cells of partially formed sheets of endoplasmic reticulum suggest that these cortical layers are newly formed by the remodelling of the endoplasmic reticulum of interphase cells. Formation of endoplasmic reticulum and chromatin-containing blebs requires both actin and microtubules, and is prevented by the caspase-6 inhibitor zVEID.fmk.

Rho kinase in the regulation of cell death and survival

“…This caspase 3-mediated ROCK1 activation is both necessary and sufficient for the formation of membrane blebs through increasing MLC phosphorylation and actomyosin contraction [16,119]. The importance of this pathway in bleb formation was then confirmed by other studies in a variety of cell types, independent of apoptotic stimuli [23,70,86,104,127,155]. In addition, ROCK1-mediated blebbing facilitates cellular fragmentation and/or apoptotic cell clearance as discussed below.…”

“…Caspase 3 is believed to be responsible for activating ROCK1 in apoptotic cells, as evidenced by the absence of ROCK1 cleavage in caspase 3-deficient MCF-7 breast carcinoma cells and the presence of ROCK1 cleavage after restoring procaspase 3 expression [119]. In addition, ROCK1 cleavage by caspase 3 can be inhibited by caspase inhibitors in a variety of apoptotic cells [16,23,70,86,104,119,127,139,140,155]. However, caspase 3-independent cleavage of ROCK1 was observed in extracellular ATP-induced and P2X7 ATP receptor-mediated apoptosis [91] and in cancer cells subjected to combined BGC9331 (a thymidylate synthase inhibitor) and SN-38 (a topoisomerase I inhibitor) treatment [17].…”

“…ROCK activation and subsequent MLC phosphorylation are required for apoptotic nuclear disintegration [18] as well as relocation of fragmented DNA into blebs and apoptotic bodies [16] in NIH3T3 cells. ROCK activity and actomyosin contraction also contribute to the formation of endoplasmic reticulum and chromatincontaining blebs in the late apoptotic cells which are likely the progenitors of apoptotic bodies [70]. Moreover, inhibition of ROCK with Y27632 prevents fragmentation of apoptotic cells and blocks Golgi fragmentation in apoptotic COS-7 and PC12 cells respectively, and these ROCK-dependent execution events require actomyosin contraction [102,103].…”

Apoptotic volume decrease as a geometric determinant for cell dismantling into apoptotic bodies

“…Indeed, membrane protrusions originated by blebbing are considered to be the progenitors of apoptotic bodies. 1,19 Blebbing (also named zeiosis) occurs in two stages. Interestingly, the first one occurs only in attached cells.…”

“…20 As a consequence, upon rounding, cell surface is in excess for the volume. 19 This type of blebbing is also seen in cells undergoing important structural rearrangements, such as division, migration, etc. 19 However, the mechanisms of this blebbing may be different from those underlying the formation of apoptotic blebs.…”