Localization sites of nuclear envelope SUN2-like proteins in root meristem cells of Allium cepa under hydroxyurea-induced DNA replication stress

Aneta Żabka,Justyna Teresa Polit,Joanna Bernasińska,Konrad Winnicki,Janusz Maszewski

doi:10.1007/s11738-015-1917-5

Abstract

Regardless of the DNA replication stress induced by low concentration of hydroxyurea (HU), root apical meristem cells of Allium cepa keep growing, and some of them override the DNA damage checkpoint mechanisms initiating either premature or an abnormal mitotic chromosome condensation. Prolonged incubation of onion seedlings with HU results in an increased level of immunodetectable proteins sharing epitopes with SUN2, one of the highly conserved elements linking nuclear envelope (NE) to the cyto- and nucleoskeletal structures. In addition to NE, phragmoplast and cell plate, our observations extend an array of subcellular compartments at which SUN2-like proteins (SUN2-LPs) are localized. These include cortical preprophase band of microtubules, centromeric regions of ana- and telophase chromosomes, and nuclear bodies (SUN2-NBs) polarly localized in interphase nuclei according to Rabl’s configuration. SUN2-NBs (distinct from fibrillarin-rich Cajal bodies) colocalize with late-replicating areas of heterochromatin and are thought to represent clustered centromeres. Three-dimensional spatial analysis of SUN2-NBs suggests their connections with NE. An enhanced expression and additional localization sites of SUN2-LPs may be correlated with a considerable reprogramming of cellular functions triggered in response to prolonged HU treatment.

Highlights

Recent experiments using long-term treatment of onion (Allium cepa) seedlings with low concentrations of HU have shown that despite persistent replication stress, a 163 Page 2 of 11Acta Physiol Plant (2015) 37:163 considerable number of root apical meristem (RAM) cells override the S-M checkpoint and initiate either premature chromosome condensation (PCC) or an unusual nuclear division forming half interphase/half mitotic chromosomes (IM cells; Z_ abka et al 2010)
Even though none of these bands matched Western blotting (WB) pattern for SUN1 protein, we found it more convenient to use the term SUN2-like proteins (SUN2-LPs) because of the unknown structure of SUN-domain proteins in A. cepa and the possible plurality of epitopic binding sites for antibodies used in our studies
Immunohistochemical and immunofluorescence labeling of the root apical meristems cells in Allium cepa indicates that most of the binding sites for the antibodies used in our experiments correspond well to the known localizations of SUN2 proteins, including nuclear membranes and two plant-specific regions, i.e. phragmoplast and the cell plate (Graumann and Evans 2011; Oda and Fukuda 2011)

Summary

Introduction

Recent experiments using long-term treatment of onion (Allium cepa) seedlings with low concentrations of HU have shown that despite persistent replication stress, a 163 Page 2 of 11Acta Physiol Plant (2015) 37:163 considerable number of root apical meristem (RAM) cells override the S-M checkpoint and initiate either premature chromosome condensation (PCC) or an unusual nuclear division forming half interphase/half mitotic chromosomes (IM cells; Z_ abka et al 2010). Recent screening studies in mammalian cell systems have demonstrated interactions between SUN-domain proteins and a conserved ATM-, ATR-, and DNA-PKmediated mechanism that function in cell cycle checkpoints to ensure genome integrity (Lei et al 2012) Considering these data, our current studies were designed to evaluate the effect of long-term HU treatment (conducted during 3 consecutive 24-h periods) on the immunolocalization of SUN2 proteins (termed here as SUN2-like proteins), which connect functionally nuclear envelope (NE) to the cyto- and nucleoskeletal structures (Evans et al 2011)

Methods

Results

Conclusion