Generation of Micronuclei during Interphase by Coupling between Cytoplasmic Membrane Blebbing and Nuclear Budding

Koh-Ichi Utani,Noriaki Shimizu,Atsushi Okamoto,Joanna Mary Bridger

doi:10.1371/journal.pone.0027233

Abstract

Micronucleation, mediated by interphase nuclear budding, has been repeatedly suggested, but the process is still enigmatic. In the present study, we confirmed the previous observation that there are lamin B1-negative micronuclei in addition to the positive ones. A large cytoplasmic bleb was found to frequently entrap lamin B1-negative micronuclei, which were connected to the nucleus by a thin chromatin stalk. At the bottom of the stalk, the nuclear lamin B1 structure appeared broken. Chromatin extrusion through lamina breaks has been referred to as herniation or a blister of the nucleus, and has been observed after the expression of viral proteins. A cell line in which extrachromosomal double minutes and lamin B1 protein were simultaneously visualized in different colors in live cells was established. By using these cells, time-lapse microscopy revealed that cytoplasmic membrane blebbing occurred simultaneously with the extrusion of nuclear content, which generated lamin B1-negative micronuclei during interphase. Furthermore, activation of cytoplasmic membrane blebbing by the addition of fresh serum or camptothecin induced nuclear budding within 1 to 10 minutes, which suggested that blebbing might be the cause of the budding. After the induction of blebbing, the frequency of lamin-negative micronuclei increased. The budding was most frequent during S phase and more efficiently entrapped small extrachromosomal chromatin than the large chromosome arm. Based on these results, we suggest a novel mechanism in which cytoplasmic membrane dynamics pulls the chromatin out of the nucleus through the lamina break. Evidence for such a mechanism was obtained in certain cancer cell lines including human COLO 320 and HeLa. The mechanism could significantly perturb the genome and influence cancer cell phenotypes.

Highlights

Growing mammalian cells often form secondary nuclei that are smaller than the main nucleus and that are referred to as micronuclei
Fusion protein, was previously established. This cell line enables the detection of double minutes (DMs) without the use of FISH, which requires heat denaturation and may disrupt the 3-D structure of the cells. Fixation of these cells by PFA followed by immunofluorescence-based detection of lamin B1 protein revealed the presence of DM-enriched micronuclei surrounded by lamina (Figure 1A) and those without lamina (Figure 1B, C), as previously reported [16,22]
The use of a cell line bearing visible DMs enabled the application of time-lapse imaging to examine the formation of DM-enriched micronuclei

Summary

Introduction

Growing mammalian cells often form secondary nuclei that are smaller than the main nucleus and that are referred to as micronuclei. Micronuclei are generated from acentric chromosomal fragments or malsegregated whole chromosomes after mitosis Such chromatin is left behind the separating chromosomes during anaphase, and generates micronuclei independently from the main nucleus at the following interphase. Acentric chromosomal fragments may be derived from unrepaired or miss-repaired chromatin after DNA double strand breakage, while malsegregated whole chromosomes can arise from chromosomes that are not bound to the spindle. The latter can occur by several mechanisms including changes in the DNA methylation level at the centromeric region The appearance of micronuclei is closely linked to the DNA damagerepair process and genome instability, and monitoring the frequency of micronuclei is widely used to assess the environmental or endogenous stresses that damage the genome and cause cancer (for a review, see the special issue of Mutagenesis, vol 26, no. 1, 2011)

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Conclusion