The biphasic interphase-mitotic polarity of cell nuclei induced under DNA replication stress seems to be correlated with Pin2 localization in root meristems of Allium cepa

5-Aminouracil and other inhibitors of DNA replication induce biphasic interphase–mitotic cells in apical root meristems of Allium cepa

“…1 B). In accordance with our earlier data (Żabka et al 2012 ; 2015a ), cell size seems to constitute one the major parameters determining either one or the other of the two abnormal types of nuclear division. The IM cell nuclei (also termed as “octopus-like” for their chromosomal arms sticking out of the interphase nuclear bodies) are restricted merely to a group to extremely long cells with major to minor axis ratios exceeding a factor of 3.0, whereas the PCC-like phenotype is confined to a group of M-phase cells approaching the maximum length to width ratio less than 2.5.…”

“…Evidence from our previous studies (Żabka et al 2010 , 2012 , 2013 , 2015a , b ) indicated that continuous treatment of onion RAMs with a low concentration of HU may disrupt crucial functions of the checkpoint response system, leading cells to abnormal mitotic progression manifested by premature chromosome condensation (PCC phenotype) and by axially elongated cells with half interphase/half mitotic structures (IM phenotype having both dispersed and condensed domains of chromatin). Our current work shows that prolonged incubations of 4-day-old primary roots of A. cepa with many chemical agents (including those commonly used to inhibit DNA replication) can induce interphase–mitotic organization of nuclear chromatin structures.…”

“…A number of side effects associated with HU-mediated inhibition of RNR comprise dissociation of the replication complex, accumulation of hemi-replicated intermediates, ssDNA interruptions at stalled RFs (which may then become converted into DSBs), formation of Holliday junctions through fork reversal, and other types of DNA damage, including those induced by ROS. Our previous studies focused on the effects of RS in onion ( Allium cepa ) primary root meristems exposed to low doses of HU clearly showed a definite interruption of the S–M checkpoint functions, leading cells either to premature chromosome condensation (PCC), or to an abnormal organization of chromatin, with both interphase (I) and mitotic (M) domains formed simultaneously on the opposing poles of the nuclei (IM cells; Żabka et al 2010 , 2012 , 2013 , 2015a , b ). Consequently, our observations were the first to demonstrate a novel means by which interphase chromatin may become gradually compacted into mitotic chromosomes, creating large-scale compartments capable to perform independent functions relevant to their specific organizational designs.…”

PIN2-like proteins may contribute to the regulation of morphogenetic processes during spermatogenesis in Chara vulgaris

“…Prior to our study on generative and non-generative antheridial cell systems, the specificity of polyclonal antibody raised against synthetic peptides derived from AtPIN2 sequences (encoded by the locus At5g57090) was tested by immunoprecipitation (IP) using total protein extracts from both apical parts of Chara thalli and root tips excised from Arabidopsis thaliana seedlings (as previously described; Żabka et al 2015 ). For each of these samples, IP followed by Western blot assay revealed two bands at about 49 and about 60 kDa (Fig.…”

“…Immunoprecipitation and protein blot assays for PIN2-LPs extracted from apical parts of C. vulgaris carrying whorls with young antheridia and (as a control) for PIN2 proteins extracted from root tips of A. thaliana (0.5–1 mm in length) were performed according to methods described earlier (Żabka et al 2015 ). Briefly, excised plant materials were lysed using a P-PER Plant Protein Extraction Kit (Pierce, Rockford, IL, USA) containing Protease Inhibitor Cocktail (P-9599; Sigma-Aldrich) and the extracts were cleared afterwards by centrifugation.…”