Biology subjects

Demidov, D.

Publications and source records attributed to Demidov, D..

4 recordsLinked to original sources

bioRxiv2025

The C-terminal SUMOylation-dependent regulation of αKNL2 governs its centromere targeting and interaction with CENH3

The centromere is a specialized domain that facilitates chromosome segregation during mitosis and serves as the site for kinetochore formation. KINETOCHORE NULL2 (KNL2) is essential for the recognition and loading of the centromeric histone H3 variant, CENH3, to centromeres. A yeast two-hybrid screen for KNL2 interactors identified components of the SUMOylation pathway. However, the role of KNL2 SUMOylation in Arabidopsis has not yet been determined. In this study, we demonstrated that the C-terminal part of KNL2 interacts with SUMO3 and ULP1d, as shown by BiFC and co-immunoprecipitation assays. Bioinformatic and functional analysis identified three SUMOylation and two SUMO-interacting motif (SIM) sites in the C-terminal region of KNL2, which are critical for growth, fertility, and chromosome alignment. Of the three SUMOylation sites, Lys474 and Lys511 were the most critical for the centromeric localization of KNL2, underscoring the importance of KNL2 SUMOylation for its function. Additionally, both in vitro and in vivo assays showed that KNL2-C undergoes SUMOylation by SUMO1 or SUMO3. The SUMO protease mutant, ulp1d-2 led to the slight accumulation of SUMOylated KNL2 in Arabidopsis. We further showed that SUMOylation of KNL2 promotes its binding to CENH3 and controls protein stability. Our findings show that C-terminal SUMOylation of KNL2 is crucial for its centromeric localization, interaction with CENH3, and kinetochore assembly, emphasizing the significance of post-translational modifications in chromosome segregation and cell division in plants.

molecular biology↗

bioRxiv2022

Plasticity in centromere organization: Holocentromeres can consist of merely a few megabase-sized satellite arrays

The centromere is the chromosome region where the microtubules attach during cell division. In contrast to monocentric chromosomes with one centromere location, holocentric species usually distribute hundreds of centromere units along the entire chromatid. We assembled the chromosome-scale reference genome and analyzed the holocentromere and (epi)genome organization of the lilioid Chionographis japonica. Remarkably, each of its holocentric chromatids consists of only 7 to 11 evenly-spaced megabase-sized centromere-specific histone H3-positive units. These units contain satellite arrays of 23 and 28 bp-long monomers capable of forming palindromic structures. Like monocentric species, C. japonica forms distinctly clustered centromeres in chromocenters at interphase. Additionally, the large-scale eu- and heterochromatin arrangement differs between C. japonica and other known holocentric species. Using polymer simulations, we modeled the formation of prometaphase line-like holocentromeres from interphase centromere clusters. Our findings broaden the knowledge about the diversity of centromere organization, showing that holocentricity is not restricted to species with numerous and small centromere units.

plant biology↗

bioRxiv2022

High temperature increases centromere-mediated genome elimination frequency in Arabidopsis deficient in cenH3 or its assembly factor KNL2

Double haploid production is the most effective way of creating true-breeding lines in a single generation. In Arabidopsis, haploid induction via mutation of the centromere-specific histone H3 (cenH3) has been shown when outcrossed to wild-type. Here we report that a mutant of the cenH3 assembly factor KNL2 can be used as a haploid inducer. We elucidated that short temperature stress of the knl2 mutant increased the efficiency of haploid induction from 1 to 10%. Moreover, we have demonstrated that a point mutation in the CENPC-k motif of KNL2 is sufficient to generate haploid inducing lines, suggesting that haploid inducing lines in crops can be identified in a naturally occurring or chemically induced mutant population, avoiding the GMO approach at any stage. In addition, we have shown that the cenh3-4 mutant, which does not induce haploids under standard growth conditions, functions as a haploid inducer after exposure to short temperature stress.

plant biology↗

bioRxiv2018

The TPX-Like protein 3TPXL, but not TPX2, is the primary activator of α Aurora kinases and is essential for embryogenesis in Arabidopsis

Aurora kinases are key regulators of mitosis. Multicellular eukaryotes generally possess two functionally diverged types. In plants like Arabidopsis, these are termed versus {beta} Auroras. As the functional specification of Aurora kinases is determined by their specific interaction partners, we initiated interactomics analyses using both Aurora kinases (AUR1 and AUR2). Proteomics results revealed the TPX2-Like proteins 2 and 3 (TPXL2/3) prominently associating with Auroras, as did the conserved TPX2 to a lower degree. Like TPX2, TPXL2 and TPXL3 strongly activated AUR1 kinase but exhibited cell cycle-dependent localization differences on microtubule arrays. The separate functions of TPX2 and TPXL2/3 were also suggested by their different influences on AUR1 localization upon ectopic expressions. Furthermore, genetic analyses disclosed that TPXL3, but not TPX2 and TPXL2, acts non-redundantly to secure proper embryo development. In contrast to vertebrates, plants expanded the TPX2 family for both redundant and unique functions among its members.

cell biology↗